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A new naphthalenone isolated from
the green walnut husks of Juglans
mandshurica Maxim.
a a a
Guang Chen , Xin-Mei Pi & Chang-Yuan Yu
a
College of Life Science and Technology, Beijing University of
Chemical Technology, Beijing 100029, P.R. China
Published online: 22 Oct 2014.
To cite this article: Guang Chen, Xin-Mei Pi & Chang-Yuan Yu (2015) A new naphthalenone isolated
from the green walnut husks of Juglans mandshurica Maxim., Natural Product Research: Formerly
Natural Product Letters, 29:2, 174-179, DOI: 10.1080/14786419.2014.971789
To link to this article: [Link]
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Natural Product Research, 2015
Vol. 29, No. 2, 174–179, [Link]
A new naphthalenone isolated from the green walnut husks of Juglans
mandshurica Maxim.
Guang Chen, Xin-Mei Pi and Chang-Yuan Yu*
College of Life Science and Technology, Beijing University of Chemical Technology, Beijing 100029,
P.R. China
(Received 3 July 2014; final version received 21 September 2014)
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Phytochemical study of green walnut husks of Juglans mandshurica Maxim. led to the
isolation of a new naphthalenone, (4R)-3,4-dihydro-4-butoxy-5-hydroxy-naphthalen-1
(2H)-one (1), together with 16 known compounds. Compounds 4 – 6, 13, 14 and 17
were isolated from the genus Juglans for the first time, and their chemotaxonomic
significance was also evaluated.
Keywords: Juglandaceae; Juglans mandshurica Maxim.; (4R)-3,4-dihydro-4-butoxy-
5-hydroxy-naphthalen-1(2H)-one
1. Introduction
Juglans mandshurica Maxim. is a deciduous tree and widely distributed in the northeast of Asia.
In addition to its edible fruit, the green walnut husks of J. mandshurica Maxim. have also been used
as folk medicines due to the recognised antioxidant, antitumour and antibacterial properties
(Fukuda et al. 2003; Deshpande et al. 2011). It has been reported that J. mandshurica Maxim. is rich
in naphthoquinones, diarylheptanoids and flavonoids (also their glycosides) (Hirakawa et al. 1986;
Lee et al. 2002; Li et al. 2007). In this study, we present the isolation and structural elucidation
of one new naphthalenone, together with 16 known compounds from green walnut husks of
J. mandshurica Maxim., and the chemotaxonomic significance of newly found constituents is also
discussed.
2. Results and discussion
Compound 1 was isolated as white amorphous powder and gave positive result with FeCl3
reagent. Its molecular formula was determined as C14H18O3 by the presence of a
pseudomolecular peak at m/z 233.1177 [M 2 H]2 in HR-ESI-MS (calcd for C14H18O2 3,
234.1257). This can also be confirmed by the distortionless enhancement by polarisation transfer
(DEPT) analysis. The hydroxyl group (3380 cm21), carbonyl group (1740 cm21) and benzene
ring (1616, 1565 and 1490 cm21) information observed in the IR spectrum of compound 1,
together with the carbonyl group (dC 197.7), benzene structure information and specific signals
at dH 2.80 –2.00 and dC 66.4, 32.5, 26.5 in its 1H NMR and 13C NMR spectra led to the
elucidation of the naphthalenone structure. In the 1H NMR spectrum of 1, the aromatic signals at
dH 7.34 (1H, d, J ¼ 7.6 Hz, H-8), 7.28 (1H, t, J ¼ 7.6 Hz, H-7) and 7.11 (1H, d, J ¼ 7.6 Hz, H-6)
showed the characteristic of AB2 spin coupling system. In addition, these three signals correlated
with dC 116.7 (C-8), 128.9 (C-7) and 120.2 (C-6), respectively, in the heteronuclear multiple
*Corresponding author. Email: yuchangy@[Link]
q 2014 Taylor & Francis
Natural Product Research 175
quantum coherence (HMQC) spectrum. In the heteronuclear multiple bond correlation (HMBC)
spectrum, a phenol proton appeared at dC 9.92 (1H, s, C-5) showed correlation peaks with dC
155.2, 129.1 and 120.2, which belonged to C-5, C-10 and C-6 positions. Since the signal at dH
7.34 (1H, d, J ¼ 7.6 Hz, H-8) also correlated with dC 197.7 (C-1), 120.2 (C-6), 128.9 (C-7) and
129.1 (C-10) in HMBC spectrum, a 4,5-dihydroxy tetralone like structure of compound 1 can be
determined. After carrying out the spectral data comparison between 1 and that of 4,5-dihydroxy
tetralone (Liu et al. 2004), a butoxy group in compound 1 was elucidated on the basis of its 13C
NMR spectrum and can be also confirmed by means of DEPT, HMQC and HMBC analyses. The
proton appeared at dH 4.86 (1H, m) showed HMQC correlation with dC 66.4 and also correlated
with dC 67.8, 129.1, 132.8 and 155.2 in HMBC spectrum. These data, together with the CH2
characteristic of dC 67.8 signal in the DEPT experiment, allowed the butoxy group to be assigned
at C-4 position. As for the absolute configuration of the chiral carbon, 4R (i.e. b-configuration)
was determined on the basis of negative Cotton effect at ca. 220 –225 nm in the CD spectrum of
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8
1 (see Supplementary material) and comparison of its optical rotation (½a20 D : 2 28 in CHCl3)
with that of literature (Machida et al. 2005).
After further comprehensive analysis of its NMR spectra (see Supplementary material),
compound 1 was identified as (4R)-3,4-dihydro-4-butoxy-5-hydroxy-naphthalen-1(2H)-one
(Figure 1), which had not been reported previously.
The known compounds were identified by comparing with the reported data as follows:
octadecane (2) (Wang et al. 2012); b-sitosterol (3) (Shen et al. 2006); stigmast-5-en-3b,7a-diol
(4) (Ren et al. 2013); stigmast-5-en-3b,7b-diol (5) (Ren et al. 2013); myricananin F (6) (Wang
et al. 2009); dibutyl phthalate (7) (Liu et al. 2007); regiolone (8) (Hirakawa et al. 1986); 4,5-
dihydroxy-a-tetralone (9) (Liu et al. 2004); olenolic acid (10) (Mahato et al. 1994); corosolic
acid (11) (Mahato et al. 1994); arjunolic acid (12) (Mahato et al. 1994); 3b,23-dihydroxy-olean-
12-en-28-oic acid (13) (Mahato et al. 1994); 3b,23-dihydroxy-urs-12-en-28-oic acid (14)
(Mahato et al. 1994); 3,4-dihydroxybenzoic acid (15) (Tang et al. 2009); daucosterol (16) (Shen
et al. 2006); 2-hydroxy-tetracosanoic acid (2,3-dihydroxy-1-hydroxymethyl-heptadec-7-enyl)-
amide (17) (Wang et al. 2010) (Figure 2).
This study led to the isolation of three naphthalenones (1, 8 and 9), one diarylheptanoid (6),
four steroids (3 –5 and 16), five triterpenoids (10 – 14), two aromatic acids (7 and 15), one
ceramide (17) and one alkane (2). Compound 1 was identified as a new naphthalenone, and
compounds 4 –6, 13, 14 and 17 were isolated from the genus Juglans for the first time.
As a unique branch of quinone compounds, naphthaquinones are widespread in the plant
kingdom and show a number of bioactivities (Kumagai et al. 2012; Nematollahi et al. 2012).
Previous review showed that naphthaquinones were detected from more than 20 families and
due to the relatively simple molecular structure, many of them play no role in terms of
chemotaxonomics (Babula et al. 2009). Although the naphthalenones are partial reduction
11 13
OH O 14 OH O
12
5 4
10
6 3
7 2
9
8 1
O O
Figure 1. Structure and key HMBC correlations of compound 1.
176 G. Chen et al.
10
HO HO OH HO OH
2 3 4 5
OH
OH O O
O
MeO OH HO O OH
OH OH
6 7 8 9
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OH OH OH OH
HO
O HO
O O O
HO
HO HO HO
OH OH
10 11 12 13
OH
O
OH H OH HO (CH2)8
OH H NH OH
O (CH2)21
HO O
OH HO O
HO H OH
OH OH OH
H H
14 15 16 17
Figure 2. Structures of compounds 2 – 17.
derivatives of naphthaquinones and share the same biosynthesis origin with naphthaquinones
(AA-MA pathway), their distribution region is much narrower than that of naphthaquinones.
However, very few naphthalenones can be used as chemotaxonomic markers (Hirakawa et al.
1986; Liu et al. 2010). In this study, the new compound 1 bears a butoxy group, which may serve
as an important structural characteristic. For example, it can be used as a chemical marker to
identify the special species among the genus of Juglans and also to distinguish Juglandaceae
from other families which are also rich in naphthaquinones.
Diarylheptanoid constituents may be more significant in chemotaxonomic research relative
to naphthaquinones. Based upon a previous review (Lv & She 2010), they are mainly distributed
in the genera of Alpinia, Zingiber, Curcuma and Alnus. Compound 6 was first isolated from
Myrica nana (Myricaceae), which belongs to cyclic-diarylheptanoids. Notably, most
diarylheptanoids isolated from the genus of Juglans belong to diphenyl ether structure type,
except for juglanin B and juglanin C from Juglans regia (Liu et al. 2008) and Juglans sinensis
(Yang et al. 2011), respectively, which belong to biphenyl structure type usually found in Myrica
genus. The findings of this study suggest that this kind of cyclic-diarylheptanoids may contribute
to systematic research.
Consistent with sphingosines and fatty acids, ceramides and their glycosylated products
called cerebrosides are widely distributed in nature. They can be easily found in animal kingdom
and considered to function as chemical messengers in the nervous system. In recent years,
numerous ceramides or cerebrosides, along with their bioactivities were reported (Tan & Chen
2003; Lynch & Dunn 2004). In the plant kingdom, ceramides/cerebrosides can be found, but
Natural Product Research 177
only in a few plant species. The first cerebroside was identified in J. mandshurica Maxim. in
2011 (Sun et al. 2011), and in this study, a ceramide compound 17 was isolated from the same
species, and this compound was only isolated from Trollius chinensis Bunge. (Wang et al. 2010)
previously. Therefore, compound 17 might serve as a chemical marker from systematic opinion.
The cytotoxic effects of isolated compounds (1 – 17) were evaluated in three human cancer
cell lines – A549, HepG2 and HT-29 – using MTT assay with 5-fluorouracil serving as the
positive control. Among these compounds, only compounds 1, 6, 8 and 9 showed cytotoxic
activity (see Table S1) in the tested cell lines. Compound 6 showed the strongest cytotoxicity in
human hepatoma HepG2 cells among the four antitumour compounds. However, this compound
exhibited weaker effect in A549 and HT-29 cells than the other three cytotoxic compounds.
Compounds 8 and 9 showed higher cytotoxicity in HepG2 and HT-29 cells than compound 1,
which showed significant cytotoxicity effect against A549 among these four compounds
indicating that the butoxy group at C-4 position may contribute to the antitumour activity.
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3. Experimental
3.1 General experimental procedure
Optical rotations were determined on a Perkin-Elmer 341 polarimeter (Perkin-Elmer, Norwalk,
CT, USA). Circular dichroism spectra were obtained on JASCO J-810-150L CD spectrometer
(Jasco, Tokyo, Japan). Bruker IFS 55 performed the IR spectrum (Bruker, Karlsruhe, Germany).
HR-ESI-MS were taken on a Bruker APEXII FT-ICR MS spectrometer (Bruker). The NMR
spectra were recorded on a Bruker 400/600 MHz (AV400/600, Berne, Switzerland) instrument
for compounds in CDCl3, CD3OD, DMSO, pyridine-d5 as solvents. Chemical shifts are
expressed in d (ppm), and are referenced to the residual solvent signals. Column
chromatography was performed using silica gel (200 –300 mesh, Branch of Qingdao Haiyang
Chemical Co., Ltd, Qindao, China). Sephadex LH-20 (GE Healthcare Bio-Sciences AB,
Uppsala, Sweden) and ODS silica gel (Merck, Darmstadt, Germany) were used for column
chromatography. TLC was performed with pre-coated silica gel GF254 glass plates (Branch of
Qingdao Haiyang Chemical Co., Ltd).
3.2 Plant material
The green walnut husks of J. mandshurica Maxim. were purchased from Anguo County of Hebei
Province, P.R. China in April 2012, and were identified by Prof. Chang-Yuan Yu, College of
Life Science and Technology, Beijing University of Chemical Technology. A voucher specimen
(No. 120411) has been deposited in the College of Life Science and Technology, Beijing
University of Chemical Technology.
3.3 Extraction and isolation
The dried green walnut husks of J. mandshurica Maxim. (15 kg) were extracted with 90% EtOH,
and the extract was resuspended in water. This suspension was then fractionated with petroleum,
ether and EtOAc, respectively. The EtOAc extract (105 g) was subjected to silica gel
chromatography (1200 g; 9 £ 100 cm column; CHCl3/EtOAc gradient) to yield nine fractions
(1 – 9). Fraction 1 was further separated by silica gel column chromatography (petroleum ether/
EtOAc 10:1) to afford 2 (5.1 mg) and 3 (8.5 mg). Fraction 2 was subjected to silica gel column
and eluted with CHCl3/acetone 20:1, 10:1 and 5:1 to yield 4 (7.3 mg) and 5 (7.5 mg). Fraction 3
was applied to passage over ODS column using MeOH/H2O (55:45) as solvent to obtain
compounds 6 (6.8 mg) and 7 (5.5 mg). Fraction 4 was further separated by Sephadex LH-20
(MeOH/H2O 45:55) to yield compounds 1 (7.1 mg), 8 (9.8 mg) and 9 (8.2 mg). Fraction 5 was
178 G. Chen et al.
subjected to ODS column and eluted with MeOH/H2O 35:65, 65:35 and 95:5 to yield
compounds 10 (7.3 mg), 11 (8.1 mg) and 12 (4.6 mg). Fraction 6 was further separated by
Sephadex LH-20 column and purified by PTLC to yield 13 (6.5 mg) and 14 (7.6 mg). Fraction 7
was purified by Sephadex LH-20 column to obtain 15 (5.6 mg). Fraction 8 was separated by ODS
column and eluted with MeOH/H2O 35:65, 65:35 and 95:5 and then purified by Sephadex LH-20
column to yield compounds 16 (5.1 mg) and 17 (9.3 mg).
3.3.1 (4R)-3,4-dihydro-4-butoxy-5-hydroxy-naphthalen-1(2H)-one (1)
8 21
White amorphous powder, ½a20 D ¼ 2 28 in CHCl3; IR (KBr, nmax, cm ): 3380, 1740, 1616,
2 2
1565, 1490; HR-ESI-MS: m/z 233.1177 [M 2 H] (calcd for C14H18O3 , 234.1257). 1H NMR
(DMSO-d6): 2.80 (1H, m, H-2a), 2.42 (1H, s, H-2b), 2.32 (1H, s, H-3a), 2.00 (1H, t, J ¼ 11.6 Hz,
H-3b), 4.86 (1H, s, H-4), 7.09 (1H, d, J ¼ 7.7 Hz, H-6), 7.27 (1H, t, J ¼ 7.7 Hz, H-7), 7.32 (1H,
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d, J ¼ 7.7 Hz, H-8), 3.36 (1H, s, H-11a), 3.60 (1H, m, H-11b), 1.45 (2H, m, H-12), 1.29 (2H, m,
H-13), 0.83 (3H, t, J ¼ 7.4 Hz, H-14), 9.92 (1H, s, 5-OH). 13C NMR (DMSO-d6): 197.7 (C-1),
32.3 (C-2), 26.5 (C-3), 66.4 (C-4), 155.2 (C-5), 120.2 (C-6), 128.9 (C-7), 116.7 (C-8), 132.8
(C-9), 129.1 (C-10), 67.8 (C-11), 31.6 (C-12), 18.8 (C-13), 11.6 (C-14).
3.4 Cell cultures
The human pulmonary adenocarcinoma cell line A549 and hepatic carcinoma cell line HepG2
were cultured in DMEM (high glucose), pH 7.3. Colorectal carcinoma cell line HT-29 was
cultured in RPMI-1640 medium, pH 7.3. Both cell cultures were supplemented with 100 IU/mL
penicillin, 100 mg/mL streptomycin and 10% foetal calf serum. Cells were grown at 378C under
5% CO2 atmosphere. Culture media were changed every 2 –3 days. When they reached
confluence, cells were dissociated by 0.05% trypsin/0.02% EDTA and re-plated at 1:5 dilutions.
3.5 MTT assay
Cancer cells were seeded onto 96-well microtitre plates at 6 £ 103 cells per well, and were pre-
incubated for 24 h at 378C. The medium was replaced with 180 mL fresh medium containing
different concentrations of each isolated compound. The cells were then incubated at 378C for
48 h, and then 20 mL of MTT was added to each well. After incubation in 378C for 4 h, the
supernatants were removed, and the formazan crystals were dissolved by adding 200 mL DMSO.
The plate was then read on a microplate reader at 490 nm to evaluate the effects of the test
compounds on cell growth. Experiments were conducted in triplicate.
Supplementary material
Supplementary material relating to this paper is available online, alongside Table S1 and
Figures S1 –S7.
Funding
This work was financially supported by the National Natural Science Foundation of China [grant number
21272023], [grant number 81273631].
References
Babula P, Adam V, Havel L, Kizek R. 2009. Noteworthy secondary metabolites naphthoquinones-their occurrence,
pharmacological properties and analysis. Curr Pharm Anal. 5:47–68.
Natural Product Research 179
Deshpande RR, Kale AA, Ruikar AD, Panvalkar PS, Salvekar JP. 2011. Antimicrobial activity of different extracts of
Juglans regia L. against oral microflora. Int J Pharm Pharm Sci. 3:200–201.
Fukuda T, Ito H, Yoshida T. 2003. Antioxidative polyphenols from walnuts (Juglans regia L.). Phytochemistry.
63:795–801.
Hirakawa K, Ogiue E, Motoyoshiya J, Yajima M. 1986. Naphthoquinones from Juglandaceae. Phytochemistry.
25:1494–1495.
Kumagai Y, Shinkai Y, Miura T, Cho AK. 2012. The chemical biology of naphthoquinones and its environmental
implications. Annu Rev Pharm Toxicol. 52:221–247.
Lee KS, Li G, Kim SH, Lee CS, Woo MH, Lee SH, Son JK. 2002. Cytotoxic diarylheptanoids from the roots of Juglans
mandshurica. J Nat Prod. 65:1707– 1708.
Li FS, Shen J, Tan GS. 2007. Research advance of chemical constituents and pharmacology activities of Juglans genus.
Chin Tradit Patent Med. 29:1490–1495.
Liu JX, Di DL, Wei XN, Han Y. 2008. Cytotoxic diarylheptanoids from the pericarps of walnuts (Juglans regia). Planta
Med. 74:754–759.
Liu LJ, Li W, Koike K, Zhang SJ, Nikaido T. 2004. New a-tetralonyl glucosides from the fruit of Juglans mandshurica.
Downloaded by [Dokuz Eylul University ] at 09:54 06 November 2014
Chem Pharm Bull. 52:566–569.
Liu Q, Zhao P, Li XC, Jacob MR, Yang CR, Zhang YJ. 2010. New a-tetralone galloylglucosides from the fresh pericarps
of Juglans sigillata. Helv Chim Acta. 93:265–271.
Liu Y, Yang X, Wu B. 2007. Chemical constituents of the flower buds of Tussilago farfara. J Chin Pharm Sci.
16:288–293.
Lv H, She G. 2010. Naturally occurring diarylheptanoids. Nat Prod Commun. 5:1687–1708.
Lynch DV, Dunn TM. 2004. An introduction to plant sphingolipids and a review of recent advances in understanding
their metabolism and function. New Phytol. 161:677–702.
Machida K, Matsuoka E, Kasahara T, Kikuchi M. 2005. Studies on the constituents of Juglans species I. Structural
determination of (4S)- and (4R)-4-hydroxy-a-tetralone derivatives from the fruit of Juglans mandshurica Maxim.
var. sieboldiana Makino. Chem Pharm Bull. 53:934– 937.
Mahato SB, Kundu AP. 1994. 13C NMR spectra of pentacyclic triterpenoids-A compilation and some salient features.
Phytochemistry. 37:1517–1575.
Nematollahi A, Aminimoqhadamfaroui N, Wiart C. 2012. Reviews on 1,4-naphthoquinones from Diospyros L. J Asian
Nat Prod Res. 14:80–88.
Ren HM, Yu CY, Yang QH, Chen H, Chen G. 2013. Chemical constituents of Senecio obtusatus. Chem Nat Compd.
49:753–754.
Shen Y, Wang QH, Lin HW, Shu W, Zhou JB, Li ZY. 2006. Study on chemical constituents of Potentilla chinensis Ser.
J Chin Med Mater. 29:237– 239.
Sun J, Chang R, Zhang H. 2011. A new cerebrogalactoside from Juglans mandshurica. Chem Nat Compd. 47:254–256.
Tan RX, Chen JH. 2003. The cerebrosides. Nat Prod Rep. 20:509–534.
Tang L, Li GY, Yang BY, Kuang HX. 2009. Study on the chemical constituents of Choerospondias axillaris. Chin Tradit
Herb Drugs. 40:541–543.
Wang JF, Zhang CL, Lu Q, Yu YF, Zhong HM, Long CL, Cheng YX. 2009. Three new diarylheptanoids from Myrica
nana. Helv Chim Acta. 92:1594–1599.
Wang RF, Liu RN, Zhang T, Wu T. 2010. A new natural ceramide from Trollius chinensis Bunge. Molecules.
15:7467–7471.
Wang YN, Cai JY, Zhao L, Zhu E, Zhang DZ. 2012. Study on chemical constituents from Anoectochilus chapaensis.
J Chin Med Mater. 35:911– 913.
Yang H, Jeong EJ, Kim J, Sung SH, Kim YC. 2011. Antiproliferative triterpenes from the leaves and twigs of Juglans
sinensis on HSC-T6 cells. J Nat Prod. 74:751– 756.