A new phenolic glycoside from Juglans mandshurica

Article abstract of DOI:10.1080/14786419.2014.902946

A new phenolic glycoside, 6-O-(4′-hydroxy-3′,5′- dimethoxybenzoyl)-d-glucopyranose (4), and nine known compounds (1-3 and 5-10) were isolated from Juglans mandshurica Maxim. Compound structures were elucidated by NMR, HR-ESI-MS and acid hydrolysis. Compounds 5 and 6 are reported from this genus for the first time. Among compounds 1-10, only 1 exhibited cytotoxicity against MGC-803, A549, K562, JAR, HeLa, CaSKi and SiHa cell lines (IC₅₀: 2.0, 5.3, 2.3, 6.9, 4.0, 6.6 and 2.7 M, respectively).

Full text of DOI:10.1080/14786419.2014.902946

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A new phenolic glycoside from Juglans
mandshurica
Dalei Yaoa, Mei Jinab, Changhao Zhanga, Jie Luoa, Ren Lib,
Mingshan Zhenga, Jiongmo Cuia & Gao Lia
a Key Laboratory of Natural Resources of Changbai Mountain and
Functional Molecules, Ministry of Education, Yanbian University
College of Pharmacy, Yanji 133002, China
b Yanbian University Hospital, Yanji 133000, China
Published online: 04 Apr 2014.
To cite this article: Dalei Yao, Mei Jin, Changhao Zhang, Jie Luo, Ren Li, Mingshan Zheng, Jiongmo
Cui & Gao Li (2014) A new phenolic glycoside from Juglans mandshurica, Natural Product Research:
Formerly Natural Product Letters, 28:13, 998-1002, DOI: 10.1080/14786419.2014.902946
To link to this article: http://dx.doi.org/10.1080/14786419.2014.902946
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Natural Product Research, 2014
Vol. 28, No. 13, 998–1002, http://dx.doi.org/10.1080/14786419.2014.902946
A new phenolic glycoside from Juglans mandshurica
Dalei Yaoa,1, Mei Jinab,1, Changhao Zhanga, Jie Luoa, Ren Lib, Mingshan Zhenga, Jiongmo Cuia
and Gao Lia*
aKey Laboratory of Natural Resources of Changbai Mountain and Functional Molecules, Ministry of
Education, Yanbian University College of Pharmacy, Yanji 133002, China; bYanbian University Hospital,
Yanji 133000, China
(Received 5 December 2013; final version received 5 March 2014)
A new phenolic glycoside, 6-O-(40-hydroxy-30,50-dimethoxybenzoyl)-D-glucopyranose
(4), and nine known compounds (1–3 and 5–10) were isolated from Juglans
mandshurica Maxim. Compound structures were elucidated by NMR, HR-ESI-MS and
acid hydrolysis. Compounds 5 and 6 are reported from this genus for the first time.
Among compounds 1–10, only 1 exhibited cytotoxicity against MGC-803, A549,
K562, JAR, HeLa, CaSKi and SiHa cell lines (IC50: 2.0, 5.3, 2.3, 6.9, 4.0, 6.6 and
2.7 mM, respectively).
Keywords: Juglans mandshurica; Juglandaceae; phenolic glycoside
1. Introduction
Juglans mandshurica Maxim. (Juglandaceae), a type of walnut tree, is mainly distributed
throughout north-eastern Asia. The roots, stem bark, leaves and fruits of this plant are used as a folk
medicine for cancer treatment in China and Korea (Xu et al. 2013). Several naphthalenyl glucosides
(Joe et al. 1996), quinones (Li et al. 2007), flavonoids (Min et al. 2002), triterpenes (Li, Lee, et al.
2003) and diarylheptanoids (Lee et al. 2002; Li, Xu, et al. 2003; Li et al. 2004, 2005) have been
isolated from J. mandshurica. These compounds have shown cytotoxic activity (Joe et al. 1996;
Lee et al. 2002; Li, Xu, et al. 2003; Li et al. 2004), topoisomerase I and II inhibitory activity
(Li, Lee, et al. 2003) and inhibitory effects on DNA polymerase and the RNase H activity of HIV-1
reverse transcriptase (Min et al. 2002). In the continuation of our studies on this plant (Lee et al.
2002; Li, Lee, et al. 2003; Li, Xu, et al. 2003; Li et al. 2004, 2005), a new phenolic glycoside (4) and
nine known compounds 1–3 and 5–10 (Figure 1) were isolated from the EtOAc solvent fraction
of the MeOH extract of J. mandshurica stem bark. Herein, we report the isolation, structural
elucidation and cytotoxic activity of these new compounds and the other constituents from the stem
bark of J. mandshurica.
2. Results and discussion
Compound 4 was obtained as white amorphous solid (MeOH). The HR-ESI-MS (negative-ion
mode) exhibited a quasi-molecular ion peak at m/z 359.0967 [M–H]– (calcd 359.0978),
corresponding to the molecular formula C15H20O10. The 1H NMR spectrum of 4 showed signals
for a galloyl group (d 7.34, s, 2H, H-20, 60), two methoxyl groups (d 3.89, s, 6H, H-30, 50-OCH3)
and a glucopyranose moiety (d 3.16–5.12, m, 7H). The a- and b-anomeric configuration
for glucopyranose was determined from JH1,H2 coupling constant value (7.3 Hz and 3.6 Hz).
*Corresponding author. Email: gli@ybu.edu.cn
q 2014 Taylor & Francis

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Figure 1. Structures of compounds 1–10 isolated from J. mandshurica.
The 13C NMR and distortionless enhanced polarization transfer spectra of 4 also showed the
presence of a glucopyranose moiety and the chemical shifts of anomeric carbon signals at d 94.0
and 98.3, corresponding to a- and b-anomeric configuration (Hsu et al. 1994), respectively. This
difference is the change in the equilibrium between two epimers of D-glucopyranose, when the
corresponding stereocentres interconvert. In the HMBC spectrum, the connectivity of the galloyl
group with glucopyranose was indicated by the cross-peak between the carbonyl group and H-6
of glucopyranose. The position of the two methoxyl groups on the aromatic ring was determined
by the HMBC correlation of C-30 with H-30-OCH3, and that of C-50 with H-50-OCH3. After acid
hydrolysis of 4, the aqueous layer was separated by HPLC to give D-glucose, and then optical
rotation of the purified glucose was determined (Kiem et al. 2003). Consequently, the structure
of 4 was identified as 6-O-(40-hydroxy-30,50-dimethoxybenzoyl)-D-glucopyranose.
The known compounds 1–3 and 5–10 were identified as, 5-hydroxy-2-methoxy-1,4-
naphthoquinone (1) (Khanna et al. 1989), 4,8-dihydroxy-1-tetralone (2) (Lal et al. 2011), 3b,24-
dihydroxyurs-12-en-28-oic acid (3) (Sashida et al. 1994), 6-O-galloyl-D-glucopyranose (5) (Hsu
et al. 1994), 1-O-galloyl-b-D-glucopyranose (6) (Yue et al. 1994), 1,2,6-trigalloylglucopyranose
(7) (Min et al. 2000), 1,2,3,6-tetragalloylglucopyranose (8) (Min et al. 2000; Duan et al. 2004),
40a,50,80-trihydroxy-a-tetralone-50-O-b-D-[6-O-(400-hydroxy-300,500-dimethoxybenzoyl)] gluco-
pyranose (9) (Kim et al. 1998) and 1,4,8-trihydroxynaphthalene-1-O-b-D-glucopyranoside
(10) (Son 1995). Compounds 5 and 6 are reported here for the first time from the genus Juglans.
Primary bioassays showed that only 1 exhibited cytotoxicity against MGC-803, A549, K562,
JAR, HeLa, CaSKi and SiHa cell lines (Table 1).
3. Experimental
3.1. General experimental procedures
Optical rotations were measured using a Rudolph Autopol I (Rudolph Research Analytical,
Hackettstown, NJ, USA) automatic polarimeter. IR spectra were recorded on a Shimadzu

1000
D. Yao et al.
Table 1. Cytotoxicity of compoundsa isolated from J. mandshurica on tumour cell growth.
IC50 (mM)
Sample
MGC-803
2.0
A549
5.3
K562
2.3
JAR
6.9
HeLa
4.0
CaSKi
6.6
SiHa
2.7
1
a Compounds 2–10 were inactive (IC50 . 100 mM).
IRPrestige-21 (Shimadzu Ltd, Tokyo, Japan) FT-IR spectrophotometer. NMR spectra were
recorded on a Bruker 600 MHz (AV-600) for 4 and on a Bruker 300 MHz (AV-300) for 1–3 and
5–10. HR-ESI-MS spectra were recorded on a Bruker microTOF QII (Bruker Daltonics,
Fremont, CA, USA) mass spectrometer. Preparative HPLC was carried out on a Bonna-Agela
FL-LC050 series chromatograph (Bonna-Agela Technologies, Tianjin, China), equipped with a
HP-Q-P050 binary pump, a Rheodyne 7725i rheodyne injector and a HP-Q-UV100 multiple
wave detector. Separations were performed on a YMC-Pack ODS-A C18 column (10 mm,
250 mm £ 10 mm, YMC, Kyoto, Japan) and a Venusil ASB C18 column (5 mm, 150 mm
£ 4.6 mm, Bonna-Agela Technologies, Tianjin, China). High-speed countercurrent chroma-
tography (HSCCC) was performed using a OptiChrome-360 HSCCC system manufactured by
Counter Current Technology Co., Ltd (Jiangyin, China), equipped with a 366 mL coil column
made of polytetrafluoroethylene tubing (i.d. 1.6 mm). Column chromatography was performed
using silica gel (200–300 mesh, Branch of Qingdao Haiyang Chemical Co., Ltd, Qingdao,
China). Sephadex LH-20 was purchased from GE Healthcare Bio-Sciences AB (GE Healthcare,
Uppsala, Sweden). TLC was performed with precoated Silica gel GF254 glass plates (Branch of
Qingdao Haiyang Chemical Co., Ltd, Qingdao, China).
3.2. Plant material
The stem bark of J. mandshurica was collected in August 2006 in a mountainous area of Yanji
City, Jilin Province, P.R. China. Plant samples were identified by Prof. Huizi Lv (College of
Pharmacy, Yanbian University). A voucher specimen (voucher number: YB-HT-0112) has been
deposited at College of Pharmacy, Yanbian University.
3.3. Extraction and isolation
The dried stem bark of J. mandshurica (10 kg) was extracted with 70% MeOH (3 £ 15 L) at
room temperature. The MeOH extract was dissolved in water and partitioned successively with
CHCl3, EtOAc and n-BuOH (each 3 £ 1 L) saturated with H2O. The EtOAc fraction (200 g) was
separated by silica gel column chromatography with a gradient of CHCl3–MeOH (100% CHCl3
to 100% MeOH) to give 15 major fractions (A–O). Fraction A (600 mg) was further applied to a
silica gel column with a gradient of CHCl3–MeOH (100:1 to 50:50), followed by reparative
HPLC with MeOH–H2O (60:40 to 90:10) to afford compounds 1 (5.5 mg), 2 (20 mg) and 3
(10.2 mg). Fractions I (500 mg) and M (630 mg) were separately purified by HSCCC using a
single solvent system composed of CHCl3–MeOH–H2O (4:3:2) to afford 4 (26 mg), 5 (10.5 mg)
and 6 (8.5 mg) from I and 9 (10.3 mg) and 10 (7.6 mg) from M. Fraction O (700 mg) was purified
by Sephadex LH-20 (MeOH–H2O, 4:1) and reparative HPLC with MeOH–H2O (20:80 to
90:10) to yield 7 (23.7 mg) and 8 (12.8 mg).
6-O-(40-Hydroxy-30,50-dimethoxybenzoyl)-D-glucopyranose (4): White amorphous solid;
25
½aꢀD 2 40.38 (c ¼ 0.03, MeOH); IR (KBr) vmax: 3407, 2924, 2852, 1714, 1564, 1464, 1342,
1222, 1114 cm–1; UV (MeOH) lmax (log 1): 277 (1.81), 223 (2.49) nm; HR-ESI-MS m/z
1
359.0967 [M–H]– (Calcd for C15H19O10, 359.0978); H NMR (CD3OD, 600 MHz) d 7.34

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(2H, s, H-20, 60), 5.12 (1H, d, J ¼ 3.6 Hz, a form H-1), 4.59 (1H, dd, J ¼ 2.0, 11.5 Hz, a and b
form H-6a), 4.53 (1H,d, J ¼ 7.3 Hz, b form H-1), 4.42 (1H, dd, J ¼ 5.5, 11.5 Hz, a and b form
H-6b), 4.10 (1/2H, m, a form H-4), 3.89 (6H, s, H-30, 50-OCH3), 3.72 (1/2H, m, a form H-3),
3.62 (1/2H, m, b form H-2), 3.48 (2H, m, a form H-2, 5 and b form H-3, 4), 3.16 (1/2H, m, b
form H-5); 13C NMR (CD3OD, 150 MHz) d 168.0 (C-70), 148.9 (C-30, 50), 142.0 (C-40), 121.2 (C-
10), 108.2 (C-20, 60), 98.3 (b form C-1), 94.0 (a form C-1), 77.9 (b form C-3), 76.2 (b form C-5),
75.5 (b form C-2), 74.7 (a form C-3), 73.8 (a form C-5), 72.1 (a form C-2), 71.8 (b form C-4),
70.9 (a form C-4), 65.3 (b form C-6), 65.2 (a form C-6), 56.8 (C-30, 50-OCH3).
3.4. Acid hydrolysis of compound 4
A solution of compound 4 (2.0 mg) in 2 mL of 5% HCl was heated for 3 h. The reaction mixture
was concentrated under reduced pressure and diluted with 15 mL H2O. The solution was
neutralised with Ag2CO3 and filtered. The aqueous layer was concentrated, filtered and passed
through a Venusil ASB C18 column (150 mm £ 4.6 mm) and then repeatedly separated by HPLC
25
(MeCN–H2O, 4:1) to afford the D-glucose fraction. The optical rotation value {½aꢀD þ 36.8
(c ¼ 0.05, H2O)} was in agreement with that of D-glucose (Crublet et al. 2003; Kiem et al.
2003).
3.5. Cytotoxicity
The tetrazolium-based colorimetric assay (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide, MTT assay) (Rubinstein et al. 1990) was used for the in vitro assay of cytotoxicity
against human gastric cancer cell line (MGC-803), human lung cancer cell line (A549), human
leukaemic cell line (K562), human placental choriocarcinoma cell line (JAR) and human
cervical cancer cell lines (HeLa, CaSKi and SiHa).
4. Conclusion
J. mandshurica is one of rare species of trees for medicinal resources. Ten compounds were
isolated from the stem bark of J. mandshurica, including one new phenolic glycoside (4) and 9
known compounds (1–3 and 5–10). 1 exhibited cytotoxicity against MGC-803, A549, K562,
JAR, HeLa, CaSKi and SiHa cell lines. The present study provides an important basis for further
research and utilisation from endangered plant species, it could help to rationalise the
conservation efforts of such plants considering that they may serve as drug leads to benefit
human health and create socioeconomic value.
Supplementary material
Supplementary material relating to this article is available online, alongside Figures S1–S15.
Acknowledgements
This work was supported by the National Natural Science Foundation of China under grant numbers
30760291, 81160386 and 30911140276.
Note
1. These authors contributed equally to the work.

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D. Yao et al.
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