Phenolic compounds from the leaves of Cyclocarya paliurus (Batal.) Ijinskaja and their inhibitory activity against PTP1B

Article abstract of DOI:10.1016/j.foodchem.2009.09.031

Phenolic compounds were separated from the leaves of Cyclocarya paliurus (Batal.) Ijinskaja and their bioactivities were evaluated through an in vitro PTP1B inhibitory assay. Bioassay-guided fractionation of the ethanol extract has resulted in the isolation of a naphthoquinone derivative, (1R, 2R, 4R)-1,2,4-trihydroxy-1,2,3,4-tetrahydro-naphthalene-1-O-β-d-glucopyranoside, named cyclonoside A, and a lactone, (4R, 5S, 6R)-8,9,10-trihydroxy-4-[3′,4′-dihydroxyphenyl]-1,6-dioxaspiro[4,5]decan-2-one, named cyclospirolide, along with 10 known phenolic compounds: quercetin-3-O-α-d-glucuronide, quercetin-3-O-β-d-glucuronide, myricetin-3-O-β-d-glucuronide, 1-caffeoylquinic acid, 3-caffeoylquinic acid, 4-caffeoylquinic acid, 5-caffeoylquinic acid, caffeic acid, 5-hydroxynaphthalene-1, 4-di-O-β-d-glucopyranoside and piceid. The structures of these compounds were established by means of spectroscopic methods including extensive 2D NMR techniques and chemical evidence. Among all the compounds, 1, 2, 4, 5, 10 and 11 showed strong inhibition against PTP1B, with IC₅₀ values ranging from 1.922 ± 0.480 to 10.50 ± 2.67 μg/mL. The results suggested that the extract from this plant could be used as a potential source for functional food ingredient with anti-obesity.

Full text of DOI:10.1016/j.foodchem.2009.09.031

Food Chemistry 119 (2010) 1491–1496
Contents lists available at ScienceDirect
Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem
Phenolic compounds from the leaves of Cyclocarya paliurus (Batal.) Ijinskaja
and their inhibitory activity against PTP1B
a,
Juan Zhang a,c, Qiang Shen b, Jin-Cai Lu c, Jing-Ya Li b, Wen-Yong Liu a, Jian-Jun Yang d, Jia Li b, , Kai Xiao
*
*
a Lab of Toxicology and Pharmacology, Faculty of Naval Medicine, Second Military Medical University, Shanghai 200433, China
b National Center for Drug Screening, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China
c School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University, Shenyang 110016, China
d Jiangxi Xiushui Shencha Co. Ltd., Xiushui, Jiangxi 332400, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 3 June 2009
Received in revised form 24 August 2009
Accepted 8 September 2009
Phenolic compounds were separated from the leaves of Cyclocarya paliurus (Batal.) Ijinskaja and their bio-
activities were evaluated through an in vitro PTP1B inhibitory assay. Bioassay-guided fractionation of the
ethanol extract has resulted in the isolation of a naphthoquinone derivative, (1R, 2R, 4R)-1,2,4-trihy-
droxy-1,2,3,4-tetrahydro-naphthalene-1-O-b-
(4R, 5S, 6R)-8,9,10-trihydroxy-4-[30,40-dihydroxyphenyl]-1,6-dioxaspiro[4,5]decan-2-one, named cyclo-
spirolide, along with 10 known phenolic compounds: quercetin-3-O- -glucuronide, quercetin-3-O-b-
-glucuronide, myricetin-3-O-b- -glucuronide, 1-caffeoylquinic acid, 3-caffeoylquinic acid, 4-caffeoyl-
quinic acid, 5-caffeoylquinic acid, caffeic acid, 5-hydroxynaphthalene-1, 4-di-O-b- -glucopyranoside
D-glucopyranoside, named cyclonoside A, and a lactone,
a
-D
Keywords:
D
D
Cyclocarya paliurus
Phenolic compounds
Naphthoquinone derivative
PTP1B inhibitors
Anti-obesity
D
and piceid. The structures of these compounds were established by means of spectroscopic methods
including extensive 2D NMR techniques and chemical evidence. Among all the compounds, 1, 2, 4, 5,
10 and 11 showed strong inhibition against PTP1B, with IC50 values ranging from 1.922 0.480 to
10.50 2.67
lg/mL. The results suggested that the extract from this plant could be used as a potential
source for functional food ingredient with anti-obesity.
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
tivity (Elchebly et al., 1999). PTP1B could also attenuate leptin
pathway, resistance to which is closely associated with obesity
(Cheng et al., 2002; Zabolotny et al., 2002). Based on these research
results, it has been suggested that components that reduce PTP1B
activity or expression levels could be used for treating obesity and
diabetes as a functional food ingredient.
In recent years, with increase of intake of foods with high en-
ergy, nutritional balance is overlooked and the incidence of obesity
and diabetes is increasing at an alarming rate. Reversible tyrosine
phosphorylation of cellular proteins plays a pivotal role for the
control of a wide range of cellular processes including the mainte-
nance of homeostasis (Alonso et al., 2004). Dephosphorylation of
the phosphorylated tyrosine residue of a protein is catalysed by
the protein tyrosine phosphatase (PTPase) family of enzymes,
which is composed of 107 highly homologous members (Alonso
et al., 2004), among which PTP1B is one of the most intensively
studied enzymes. There is substantial evidence that PTP1B is the
critical point in the insulin signalling pathway (Bialy & Waldmann,
2005; Johnson, Ermolieff, & Jirousek, 2002; Koren & Fantus, 2007;
Kraut-Cohen, Muller, & Elson, 2008). The overexpression of PTP1B
has been shown to inhibit the increased expression of insulin in
insulin-resistant states (Ahmad, Azevedo, Cortright, Dohm, & Gold-
stein, 1997). Furthermore, recent genetic evidence has shown that
PTP1B gene variants are associated with changes in insulin sensi-
Phenolic compounds have shown beneficial bioactivities, such
as antioxidant (Peterson & Dwyer, 1998), anticarcinogenic (Bailey
& Williams, 1993), antibacterial (Meng, Lozano, Bombarda, Gay-
dou, & Li, 2008), antimutagenic (Liverio, Puglisi, Morazzoni, & Bom-
bardelli, 1994), anti-inflammatory (Ueda, Yamazaki, & Yamazaki,
2002), antiallergic (Sanbongi et al., 2004), anti-obesity and anti-
diabetic activities (Hsu & Yen, 2008). Phenolic compounds, espe-
cially flavonoids, in the light of their extremely high values, have
become a hot topic for research and development. Consequently,
phenolic compounds are not only used as functional food ingredi-
ents, but also for other preparations of health-promoting products.
Traditional Chinese medicines are good sources of phenolic
compounds and have a long history and are used in the treatment
of obesity and diabetes and as a potential source for functional
food ingredients. Therefore, we have screened hundreds of plant
extracts against this biological target, and the extract of Cyclocarya
paliurus (Batal.) Ijinskaja showed strong inhibitory activity against
* Corresponding authors. Tel.: +86 21 50801313x132; fax: +86 21 50801552 (J.
Li), tel.: +86 21 81871129; fax: +86 21 81871128 (K. Xiao).
PTP1B (IC50 = 1.27
lg/mL). C. paliurus belongs to the genus
E-mail addresses: jli@mail.shcnc.ac.cn (J. Li), kaixiaocn@gmail.com (K. Xiao).
0308-8146/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodchem.2009.09.031

1492
J. Zhang et al. / Food Chemistry 119 (2010) 1491–1496
Cyclocarya (Juglandaceae), which is mainly distributed in southern
provinces of China. C. paliurus has been used as a traditional tonic
and its leaves are processed as tea products in China. Previous
chemical studies have reported the presence of abundant phenolic
compounds, especially flavonoids, from Cyclocarya species (Ager-
strand, Ruden, & Wester, 2008; Burge & Manchester, 2008; Li
et al., 2008). Thus, it is important to develop C. paliurus as a func-
tional food or an ingredient to be used for treatment of diabetes
and obesity.
In the present work, water-soluble extract of the leaves of C.
paliurus was used for bioassay-guided fractionation leading to
the isolation of 12 secondary metabolites, two of which were novel
structures. The compounds obtained were determined for their
inhibitory activity against PTP1B.
on TSK gel Toyopearl HW40F and Cosmosil ODS (40–80 lm, Naca-
lai Tesque Inc., Kyoto, Japan) repeatedly, and eluted with water to
afford compounds 6 (35 mg), 7 (25 mg), 8 (24 mg) and 9 (26 mg).
Fraction B (IC50 = 2.95 0.60 lg/mL) was separated by polyamide
(80–100 mesh) using a gradient of ethanol (from 10% to 95%), to
yield six sub-fractions (sub-fractions B-1–B-6). Further repeated
purification of sub-fractions B-2 (78 mg), B-3 (87 mg) and B-5
(67 mg) by column chromatography on TSK gel Toyopearl
HW40F, using water as eluant, resulted in the isolation of com-
pounds 3 (24 mg), 4 (15 mg) and 5 (22 mg), respectively. Fraction
C (IC50 = 3.57 0.32 lg/mL) was separated by column chromatog-
raphy on TSK gel Toyopearl HW40F using a stepwise gradient to
yield five sub-fractions (sub-fractions C-1–C-6). Then sub-fractions
C-1 and C-2 were purified by repeated column chromatography on
TSK gel Toyopearl HW40F and Cosmosil ODS, using water as elu-
ant, to afford compounds 1 (47 mg) and 2 (7 mg). Fraction D
2. Materials and methods
(IC50 = 3.47 0.39 lg/mL) was separated by MCI gel CHP20P, using
a gradient of methanol (from 10% to 60%), to give colourless and
transparent crystal. Through repeated recrystallization, compound
12 (34 mg) was obtained.
2.1. Plant material
The leaves of C. paliurus were collected in March 2004 at Xiu-
shui in the Jiangxi Province of China and identified by Jianjun Yang
of Jiangxi Xiushui Shencha Co. Ltd. A voucher specimen has been
deposited at Lab of Toxin and Pharm, Faculty of Naval Medicine,
Second Military Medical University, Shanghai, China.
2.3.1. Cyclonoside A (1)
MeOH
max
Colorless and crystal needles, m.p. 156–157 °C. UV k
nm
(log
e
): 212 (2.30) and 261 (0.07). IR mMmaexOH: 3428, 3343, 2922,
2852, 1700, 1651, 1558, 1456, 1379, 1322, 1259, 1162, 1022,
989, 930 and 892 cmꢀ1 1H and 13C NMR (see Table 1). ESI-TOF-
.
2.2. General experimental procedures
MS+ m/z 365.91 [M+Na]+, 707.48 [2M+Na]+, 804.89, 533.24,
464.07, 414.22, 360.59, 260.48; ESI-TOF-MSꢀ m/z 341.50 [MꢀH]ꢀ,
683.11 [2MꢀH]ꢀ, 477.28, 387.34. HRESI-MS m/z 365.1214 (calcd.
for C16H22O8 + Na, 365.1212).
All melting points were determined on a SGW X-4 melting point
apparatus (Shanghai Huayan Instrument Co. Ltd., Shanghai, China).
The UV spectra were obtained on a CARY100 UV–Vis Spectropho-
tometer (Varian Inc., Palo Alto, CA, USA). The IR spectra were re-
2.3.2. Acid hydrolysis of 1
corded on
a Vector 22 Infrared Spectrophotometer (Bruker
Corporation, Bremen, Germany). The 1H, 13C NMR and 2D NMR
data (including HMQC, HMBC, NOESY and 1H–1H COSY) were mea-
sured on a Bruker AV-500 spectrometer instrument (Bruker Corpo-
ration, Bremen, Germany) operating at 500 MHz for 1H and
125 MHz for 13C. The chemical shifts (d) are reported in ppm down-
field from tetramethylsilane (TMS) using TMS or the solvent signal
as standard. The HRESI-TOF-MS and ESI-TOF-MS/MS were obtained
on a Q-TOF micromass spectrometer (Waters Corporation, Man-
chester, England) and LCQ Deca XP Max Liquid Chromatography-
Mass (Thermo Scientific Co. Ltd., New York, USA). Analytical thin
layer chromatography (TLC) was performed on HSGF254 and the
spots were detected by ultraviolet irradiation (254 and 365 nm)
and by spraying with vanillin-H2SO4 reagent.
About 2 mg of 1 was refluxed in 2 mL 7% HCl–EtOH (3/7) for 4 h.
The mixture was diluted with distilled water and extracted with
diethyl ether. The aqueous layer was neutralised with 1 M NaOH
and subjected to TLC analysis on Kieselgel 60 F254 (Merck Chemical
Co. Ltd., Darmstadt, Germany) [using CHCl3–MeOH–H2O (15/6/2),
9 mL and HOAc, 1 mL] and paper chromatography [using n-
BuOH–HOAc–H2O (4/1/5)] with standard sugars, in this case the
presence of glucose was established. The aqueous layer was then
passed through an Amberlite IRA-60E column (Sigma–Aldrich Co.
Ltd., St. Louis, MO, USA), and the aqueous eluate was concentrated
and treated with thiazolidine. Only the D-glucose derivative was
detected by GC (GC conditions: column, Supelco SPBꢀ1
,
Table 1
1H NMR (500 MHz, DMSO-d6 + D2O), 1H NMR (500 MHz, DMSO-d6) and 13C NMR
(125 MHz, DMSO-d6 + D2O) of compound 1.
2.3. Extraction and isolation
The dried leaves (10 kg) of C. paliurus were extracted with 70%
ethanol under reflux condition three times. The suspension was fil-
tered and the filtrates were concentrated under in vacuum to give
No.
dH (DMSO-d6 + D2O)
dH (DMSO-d6)
dC
1
2
4.41 (1H, d, J = 6.3)
4.08 (1H, m)
1.91 (1H, m)
4.40 (1H, d, J = 6.3)
4.07 (1H, m)
1.90 (1H, m)
82.7 (d)
67.1 (d)
36.6 (t)
crude extract (IC50 = 1.27
column chromatography on Diaion HP20 macropore polymeric
adsorbent (200–600 m, Japan Mitshubishi Chemical Corporation,
lg/mL). This residue was subjected to
3a
3b
4
5
6
7
2.00 (1H, m)
1.99 (1H, m)
4.69 (1H, t-like, J = 5.1)
7.38 (1H, br d, J = 7.3)
7.27 (1H, dt, J = 7.3, 1.3)
7.23 (1H, dt, J = 7.3, 1.3)
7.67 (1H, br d, J = 7.3)
–
4.68 (1H, q-like, J = 5.2)
7.37 (1H, br d, J = 7.3)
7.26 (1H, dt, J = 7.3, 1.2)
7.21 (1H, dt, J = 7.3, 1.2)
7.68 (1H, br d, J = 7.3)
–
65.1 (d)
128.0 (d)
127.6 (d)
127.2 (d)
128.9 (d)
134.9 (s)
139.7 (s)
103.6 (d)
73.8 (d)
76.9 (d)
70.3 (d)
76.8 (d)
61.2
l
Tokyo, Japan), after removing sugar with water elution, using a
stepwise gradient of ethanol (10–90%) to give four fractions la-
belled A (105 g), B (108 g), C (68 g) and D (117 g) according to their
8
9
TLC profiles. Fraction A (IC50 = 4.10 0.15
column chromatography on MCI gel CHP20P (75–150
l
g/mL) was separated by
m, Japan
10
10
20
30
40
50
–
–
l
4.45 (1H, d, J = 7.8)
3.10 (1H, m)
3.20 (1H, m)
3.20 (1H, m)
3.10 (1H, m)
4.44 (1H, d, J = 7.8)
3.08 (1H, m)
3.18 (1H, m)
3.18 (1H, m)
3.08 (1H, m)
Mitsubishi Chemical Corporation, Tokyo, Japan), eluted with meth-
anol (10–90%) gradiently to yield three sub-fractions (sub-fractions
A-1–A-3). Sub-fraction A-1 was again subject to column chroma-
tography on TSK gel Toyopearl HW40F (30–60 lm, Tosoh Co.
Ltd., Tokyo, Japan) and eluted with water to afford compound 10
6
0a
3.75 (1H, dd, J = 11.6, 1.5)
3.46 (1H, dd, J = 11.6, 6.7)
3.74 (1H, m)
3.45 (1H, m)
60b
(36 mg). Sub-fraction A-3 was purified by column chromatography

J. Zhang et al. / Food Chemistry 119 (2010) 1491–1496
1493
0.25 mm ꢁ 27 m (Sigma–Aldrich Co. Ltd., St. Louis, MO, USA), col-
umn temperature 230 °C; carrier gas, N2; tR, -glucose derivative
17.9 min, -glucose derivative 17.3 min).
For calculating IC50, inhibition assays were performed with
30 nM recombinant enzyme, 2 mM pNPP in 50 mM MOPS at pH
6.5 and the inhibitors diluted around the estimated IC50 values.
IC50 was calculated from the non-linear curve fitting of per cent
inhibition (% inhibition) vs. inhibitor concentration I by using the
following equation:
D
L
2.3.3. Cyclospirolide (2)
MeOH
max
Amorphous powder, m.p. 156–157 °C. UV k
nm (log e): 205
(1.82) and 282 (0.23). IR mMmaexOH: 3479, 3425, 3194, 3038, 2984,
% Inhibition ¼ 100=f1 þ ðIC50=½IꢂÞkg
where k is the Hill coefficient.
2931, 2898, 1805, 1787, 1677, 1604, 1279, 1260, 1222, 1200,
1137, 1122, 1075, 1052, 1028 and 967 cmꢀ1 1H and 13C NMR
.
(see Table 2). ESI-TOF-MSꢀ m/z 311.36 [MꢀH]ꢀ, 623.32 [2MꢀH]ꢀ,
679.45, 401.14, 347.16, 221.33, 177.36. HRESI-MS m/z 312.0827
(calcd. for C14H16O8, 312.0845).
2.5. Statistical analysis
For bioactivity assay, all determinations were triplicates, and
mean values and standard deviations were calculated. Analysis of
variance (ANOVA) was performed and the mean separation was
done by LSD (P 6 0.05) using SPSS 13.0 program for windows (SPSS
Inc., IL, USA).
2.4. Bioactivity assay
2.4.1. PTP1B enzymatic assay
PTP1B (human, recombinant) was expressed and purified, and
the enzyme activity was measured at 30 °C by monitoring the
hydrolysis of pNPP (Zhang et al., 2006). The enzymatic activities
of PTP1B catalytic domain were determined at 30 °C by monitoring
the hydrolysis of pNPP. Dephosphorylation of pNPP generates
product pNP, which can be monitored at 405 nm. In a typical
3. Results and discussion
3.1. Extraction and isolation
Bioactivity-guided fractionation of the water-soluble extract of
C. paliurus, using an in vitro PTP1B inhibitory assay, led to the iso-
lation and characterisation of a new naphthoquinone derivative,
named cyclonoside A (1), and a new lactone named cyclospirolide
(2), along with 10 known secondary metabolites (3–12). The
known compounds were identified by comparison of their physico-
chemical and spectroscopic data (1H, 13C NMR, 2D NMR and MS)
with those of authentic samples and reference data as: querce-
100 lL assay mixture containing 50 mM MOPS, pH 6.5, 2 mM pNPP
and recombinant enzymes, PTP1B activities were continuously
monitored and the initial rate of the hydrolysis was determined
using the early linear region of the enzymatic reaction kinetic
curve.
2.4.2. PTP1B inhibition screening
The samples were screened against the PTP1B with the colori-
metric assay. The high-throughput screening system of PTP1B
was similar with LAR (Zhang et al., 2006). Briefly, the samples were
tin-3-O-
a
-D
-glucuronide (3), quercetin-3-O-b-
D-glucuronide (4),
myricetin-3-O-b-
D-glucuronide (5) (Loke et al., 2008; Nakanishi
et al., 2007), 1-caffeoylquinic acid (6), 3-caffeoylquinic acid (7),
4-caffeoylquinic acid (8), 5-caffeoylquinic acid (9), caffeic acid
(10) (Liu et al., 2009; Zhou et al., 2009), 5-hydroxynaphthalene-1,
4-di-O-b-D-glucopyranoside (11) (Liu, Li, Koike, & Nikaido, 2004)
and piceid (12) (Fig. 1) (Jayatilake et al., 1993).
solubilised in dimethyl sulphoxide at 5 mg/mL, and 2
were distributed to A2–H11 wells of 96-well clear polystyrene
plate (Corning, Action, MA). The dimethyl sulphoxide (2 L) was
distributed to A1–D1 and E12–H12 wells as the full enzyme activ-
ity, and orthovanadate (12.5 mM, 2 L) was distributed to E1–H1
and A12–D12 wells as the positive inhibition. After adding an assay
mixture (88 L), 10 L of the GST-PTP1B (300 nM) were added to
initiate the reaction. The high-throughput screening was carried
out in a final 100 L volume containing 50 mM MOPS, pH 6.5,
lL samples
l
l
3.2. Structure determination of compound 1
l
l
Compound 1, m.p. 155–157 °C, was obtained as colourless and
crystal needles from a 10% ethanol solution. It showed positive re-
sults in Molish test, suggesting the presence of sugar moiety in the
molecule. Its molecular formula was determined to be C16H22O8
using positive high-resolution electrospray ionisation time of flight
(HR-ESI-TOF) mass spectroscopy which showed a quasi-molecular
ion [M+Na]+ peak at m/z 365.1214 (calcd. for C16H22O8Na,
365.1212). The ESI-MS spectrum in negative ion mode exhibited
the quasi-molecular ion [MꢀH]ꢀ peak at m/z 341.50 and ion
[2MꢀH]ꢀ peak at m/z 683.11. The IR spectrum of compound 1 dis-
l
2 mM pNPP, 30 nM PTP1B and 2% dimethyl sulphoxide, and the
catalysis of pNPP was continuously monitored on SpectraMax
340 microplate reader (Molecular Devices Corporation, Sunnyvale,
USA) at 405 nm for 2 min at 30 °C.
Table 2
1H NMR (500 MHz, DMSO-d6 + D2O), 1H NMR (500 MHz, DMSO-d6) and 13C NMR
(125 MHz, DMSO-d6 + D2O) of compound 2.
No.
2
dH (DMSO-d6 + D2O)
dH (DMSO-d6)
dC
played the presence of hydroxyl group
3428.33 cmꢀ1) and aromatic ring (mmax 1455.17–1558.94 cmꢀ1
(Wang, Liu, Liu, Zhang, & Xian, 2008), which was also evidenced
by the UV spectrum [kmax (log
): 261 (0.07) nm]. The 1H NMR
(mmax 3343.45–
)
–
–
175.3 (s)
36.6 (t)
3a
2.68 (1H, dd, J = 9.2, 17.8)
2.96 (1H, dd, J = 9.2,17.8)
3.74 (1H, t, J = 9.2)
–
3.27 (1H, t, J = 9.8)
3.62 (1H, dd, J = 5.3, 9. 8)
3.57 (1H, dd, J = 5.4, 9.4)
3.51 (1H, dd, J = 2.5, 9.4)
3.79 (1H, overlapped)
–
6.71 (1H, s)
–
–
6.62 (1H, d, J = 8.0)
6.57 (1H, d, J = 8.0)
2.63 (1H, dd, J = 9.0, 17.8)
2.95 (1H, dd, J = 9.0, 17.8)
3.72 (1H, t, J = 9.0)
–
3.25 (1H, t, J = 9.6)
3.58 (1H, overlapped)
3.58 (1H, overlapped)
3.49 (1H, br s)
3.77 (1H, br.s)
–
6.68 (1H, s)
–
–
6.64 (1H, d, J = 8.0)
6.61 (1H, d, J = 8.0)
3b
4
5
e
46.6 (d)
109.2 (s)
65.8 (t)
spectrum (DMSO-d6 + D2O) (Table 1) of compound 1 exhibited
two double doublets at dH 7.67 (1H, dd, J = 7.3, 1.3 Hz) and dH
7.38 (1H, dd, J = 7.3, 1.3 Hz) and two double triplets at dH 7.27
(1H, dt, J = 7.3, 1.3 Hz), dH 7.23 (1H, dt, J = 7.3, 1.3 Hz), which were
assignable to a 1,2-disubstituted benzene ring with the assistance
from the analysis of HMBC (Fig. 2A1) and HMQC experiments. The
13C NMR spectrum (Table 1) of compound 1 revealed 16 carbon
signals as two methylenes, 12 methines and two quaternary car-
bons assignable to one benzene ring, one glucose, three oxygen-
ated sp carbons and one sp2 carbon. Full analysis of the COSY,
HMBC and HMQC spectra led to the identification of a 1,2,3,4-tet-
7
a
7b
8
9
10
10
20
30
40
50
60
65.5 (d)
71.6 (d)
72.9 (d)
117.4 (d)
144.4 (s)
144.2 (s)
115.1 (d)
121.6 (d)
129.2 (s)

1494
J. Zhang et al. / Food Chemistry 119 (2010) 1491–1496
HO
O
6'
4'
5'
2'
O
O
2
3
HO
H
HO
1'
H
4
O
1'
OH
3'
10
OH
OH
1
OH
H
5
9
7
H
8
2
4'
7
3
O
HO
3'
4
8
6
OH
6
OH
5
H
H
OH
1
2
R1
OH
O
OH
OH
R4
HO
HO
O
OR
R3
HO
O
R1
R2
R2
6
7
8
9
R = R1, R2 = OH, R3 = OH, R4 = OH
R = R2, R1 = OH, R3 = OH, R4 = OH
R = R3, R1 = OH, R2 = OH, R4 = OH
R = R4, R1 = OH, R2 = OH, R3 = OH
OH
O
3 R1 = H, R2 = 3-O-α-D-glucuronic acid
4 R1 = H, R2 = 3-O-β-D-glucuronic acid
5 R1 = OH, R2 = 3-O-β-D-glucuronic acid
10 R = H
HO
OH
O
O
O
HO
HO
OH
O
HO
O
HO
OH
HO
HO
OH
HO
OH
O
OH
OH
12
11
Fig. 1. Chemical structures of compounds 1–12.
HO
HO
O
O
O
O
HO
HO
HO
OH
H
HO
H
OH
H
H
H
H
OH
OH
H
H
H
H
H
H
H
H
H
OH
H
OH
A1
A2
H
H
H
O
O
O
H
H
H
H
H
H
O H
O
H
H
H
OH
OH
OH
OH
H
H
O
OH
HO
OH
H
HO
H
H
H
H
OH
OH
B1
B2
Fig. 2. Significant HMBC (1) and NOESY (2) correlations of compound 1 (A1 and A2) and 2 (B1 and B2).
rahydronaphthalene skeleton as shown in Fig. 1. The 13C NMR pat-
tern of the sugar moiety was characteristic of a glucose (Agrawal,
ase, Saitoh, Shiokawa, & Ueno, 1995). In the HMBC of compound 1,
the significant correlations between H0-1 (d 4.45) and C-1 (d 82.7),
H-1 (d 4.41) and C-1 (d 103.6) indicated that the glucose was at-
tached to C-1, and the b-linkage of the glucose was determined
1992) and was further determined as
D-glucose according to the re-
sults of the acidic hydrolysis and the subsequent GC analysis (Miy-

J. Zhang et al. / Food Chemistry 119 (2010) 1491–1496
1495
from the coupling constant of the anomeric proton (J = 7.8 Hz)
(Agrawal, 1992). A combination analysis of the HMQC, HMBC and
1H–1H COSY experiments allowed the unambiguous assignment
of all the signals (Table 1). Moreover, in the NOESY spectrum of
determined to be C14H16O8 as evidenced by HR-ESI-TOF mass spec-
troscopy which showed a molecular ion [M]+ peak at m/z 312.0827
(calcd. for C14H16O8, 312.0845). The ESI-MS spectrum in negative
ion mode showed the quasi-molecular ion [MꢀH]ꢀ peak at m/z
311.36 and ion [2MꢀH]ꢀ peak at m/z 623.32. The IR spectrum of
2 suggested the presence of hydroxyl group (mmax 3194.24–
compound 1, strong correlations between H-3
(d 4.69), and between H-3b (d 2.00) and H-2 (d 4.08) showed that
H-1, H-3 and H-4 located at the same side of the planar, while
a (d 1.91) and H-4
a
3479.68 cmꢀ1), carbonyl group ( max 1787 cmꢀ1) and aromatic ring
m
H-2 and H-3b located at the opposite side (Fig. 2A2).
(m
max 1416.78–1604.25 cmꢀ1). The UV spectrum also suggested the
existence of benzene ring and carbonyl group in compound 2 [kmax
(log ): 282 (0.24) nm] (Kenawi, Barsoum, & Youssef, 2005). In the
Finally, the structure and stereochemistry of compound 1 were
further established by means of X-ray single-crystal crystallography
analysis (Fig. 3). Crystal data of compound 1 was exhibited as
follows: C16H24O9 (co-crystallized with one H2O molecules), Mr
360.35, monoclinic, space group P21, a = 8.1569(10) Å, b =
12.4319(15) Å, c = 8.3561(10) Å, b = 100.559(2)°, V = 833.01(17) Å3,
e
1H NMR spectrum (DMSO-d6 + D2O) (Table 2) of 2, an ABC spin sys-
tem, resonating at dH 6.62 (1H, d, J = 8.0 Hz), 6.57 (1H, d, J = 8.0 Hz)
and 6.71 (1H, s), could be attributed to a 1,3,4-trisubstituted ben-
zene ring. The 13C NMR spectrum (Table 1) of compound 2 showed
14 carbon signals as two methylenes, seven methines and five qua-
ternary carbons, among which one carbonyl group (d 175.3), one
benzene ring and five oxygen-bearing carbons [d 65.5 (d), 65.8
(t), 71.6 (d), 72.9 (d) and 109.2 (s)] could be assigned. Though com-
pound 2 exhibited some resemblance to the NMR pattern of sugar
moiety, negative result in Molish test excluded the presence of su-
gar moiety in the molecule. In addition of the benzene ring, full
analysis of the 1H, 13C NMR, COSY, HMBC and HMQC spectra re-
Z = 2, Dc = 1.437 Mg mꢀ3
,
F(0 0 0) = 536, k = 0.71073 Å,
l =
0.118 mmꢀ1. Data were collected from a 0.20 ꢁ 0.15 ꢁ 0.15 mm3
crystal on a Bruker SMART APEX CCD area detector diffractometer
at 296(2) K.
A total of 5225 reflections were collected for
2.48° < h < 27.00° and ꢀ10 6 h 6 9, ꢀ12 6 k 6 15, ꢀ10 6 l 6 10.
There were 1904 independent reflections. Semi-empirical absorp-
tion correction from equivalents was applied. The structure was
solved by means of direct method with SHELXS-97 to final indices
R
[F2 > 2
[w = 1/[
r
(F2): 1904 reflections] = 0.0293 and wR(F2) = 0.0740,
vealed the presence of one c-bytyrolactone group [dH 3.74/dC
r
2(F2a)+(0.053P)2], where P = (Fo2 + 2Fc2)/3]. The hydrogen
46.6 (1H, t, J = 9.2), two double doublets at dH 2.96/dC 36.6 (1H,
dd, J = 9.2, 17.8), dH 2.68/dC 36.6 (1H, dd, J = 9.2, 17.8), dC 175.3
and 109.2], one saturated trihydroxypyran ring moiety and one
lactol ether linkage, which formed 1,6-dioxaspiro[4,5]decan-2-
one as the skeleton of compound 2. Analysis of the 1H–1H COSY,
HMQC and HMBC experiment results permitted the full assign-
ment of the signals (Table 2). Furthermore, the coupling constants
of proton signals on the trihydroxypyran ring demonstrated that
the hydroxyl groups of compound 2 were axially-oriented at C-
10 and equatorial at C-9 and C-8 in a chair conformation. In the
NOESY spectrum of 2, significant correlations between H-4 (d
atoms were placed in calculated positions and added to the refine-
ment as a fixed isotropic contribution. The goodness-of-fit on F2
was 1.057, and the last residual Fourier positive and negative peaks
were equal to 0.196 and ꢀ0.198, respectively. Crystallographic data
(excluding structure factors) for the structure in this paper have
been deposited with the Cambridge Crystallographic Data Centre
as supplementary publication numbers CCDC 721236. Copies of
the data can be obtained free of charge on application to CCDC, Cam-
bridge, UK. Since the absolute configuration of the sugar has been
established, the absolute structure of compound 1 has also resolved
in the X-ray analysis. On the basis of the above results, the structure
of compound 1 was determined to be (1R, 2R, 4R)-1,2,4-trihydroxy-
3.74) and H-3
showed that H-4 and H-3
a
(d 2.96), between H-9 (d 3.51) and H-7b (d 3.27)
located at the same planar of the
a
c-
1,2,3,4-tetrahydro-naphthalene-1-O-b-
cyclonoside A.
D
-glucopyranoside, named
bytyrolactone ring, and H-9 and H-7b located at the same planar
of the saturated trihydroxypyran ring, respectively (Fig. 2B2). It
turned out that compound 2 was a derivative of sawaranospirolide
D. Compared to sawaranospirolide D reported by Hasegawa et al.
(1990), the benzene ring of 2 possessed one more additional hy-
droxyl group, and the rest parts of compound 2 were in agreement
with sawaranospirolide D. On the basis of the results described
above, the structure of compound 2 was determined to be (4R,
5S, 6R)-8, 9, 10-trihydroxy-4-[30,40-dihydroxyphenyl]-1,6-dioxa-
spiro[4,5]decan-2-one (Fig. 1), named cyclospirolide.
3.3. Structure determination of compound 2
Compound 2, m.p. 291–292 °C, was obtained as amorphous
powder from a 10% ethanol solution. Its molecular formula was
3.4. Determination of inhibitory activity against PTP1B
PTP1B (human, recombinant) was expressed and purified, and
the enzyme activity was measured using p-nitrophenyl phosphate
(pNPP) as a substrate (Burke et al., 1996). All the isolates were as-
sayed for their inhibitory activity against PTP1B, and the results
were presented in Table 3. The known PTP1B inhibitor HD0518
was used as positive controls in this assay. The bioactivity in the
total extract was higher than in any fraction obtained using 10–
90% ethanol as eluent, suggesting that interaction effect among dif-
ferent compounds might contribute to inhibitory effect against
PTP1B. Six compounds inhibited PTP1B activity in a dose-depen-
dent manner. Of the compounds tested, 1 and 10 exhibited the
most potent inhibitory activities, with IC50 values of 2.11 0.66
and 1.922 0.480
Compound 5 (IC50 = 9.47 3.31
xyl group substituted at C-50, was a little less active than com-
pound 4 (IC50 = 7.39 1.15 g/mL), indicating that addition of
one hydroxyl group to C-50 in the B ring may be responsible for
l
g/mL, respectively.
lg/mL), with one more hydro-
l
Fig. 3. ORTEP drawing of compound 1.

1496
J. Zhang et al. / Food Chemistry 119 (2010) 1491–1496
Bialy, L., & Waldmann, H. (2005). Inhibitors of protein tyrosine phosphatases: Next-
Table 3
generation drugs? Angewandte Chemie, International Edition in English, 44(25),
3814–3839.
Burge, D. O., & Manchester, S. R. (2008). Fruit morphology, fossil history, and
biogeography of paliurus (rhamnaceae). International Journal of Plant Sciences,
169(8), 1066–1085.
Burke, T. R., Ye, J. B., Yan, X., Wang, S., Jia, Z., Chen, L., et al. (1996). Small molecule
interactions with protein–tyrosine phosphatase PTP1B and their use in inhibitor
design. Biochemistry, 35(50), 15989–15996.
Cheng, A., Uetani, N., Simoncic, P. D., Chaubey, V. P., Lee-Loy, A., McGlade, C. J., et al.
(2002). Attenuation of leptin action and regulation of obesity by protein
tyrosine phosphatase 1B. Developmental Cell, 2(4), 497–503.
Elchebly, M., Payette, P., Michaliszyn, E., Cromlish, W., Collins, S., Loy, A. L., et al.
(1999). Increased insulin sensitivity and obesity resistance in mice lacking the
protein tyrosine phosphatase-1B gene. Science, 283, 1544–1548.
Hasegawa, S., Koyanagi, H., & Hirose, Y. (1990). Decarboxylated ascorbigens in the
heartwood of Chamaecyparis pisifera. Phytochemistry, 29(1), 261–266.
Hsu, C. L., & Yen, G. C. (2008). Phenolic compounds: Evidence for inhibitory effects
against obesity and their underlying molecular signaling mechanisms.
Molecular Nutrition and Food Research, 52, 53–61.
Inhibitory activity of different fractions and compounds 1–12 against PTP1B.
Fraction
IC50
(l
g/mL)A,B,C
Compounds
IC50 (l
g/mL)A,B,C
Total extract
Fraction A
Fraction B
Fraction C
Fraction D
1.27 0.12a
4.10 0.15b
2.95 0.60c
3.57 0.32d
3.47 0.39e
1
2
4
5
10
11
2.11 0.66a
16.64 0.04b
7.39 1.15c
9.47 3.31a
1.922 0.480d
10.50 2.67e
Means within columns with different superscripts were significantly different
(P 6 0.05).
A
IC50 values were determined by regression analyses and expressed as
mean SD of three replicates.
B
Compounds 3–9 were inactive.
Positive control HD0518 with IC50 value of 1.12 0.18 lg/mL.
C
Jayatilake, G., Jayasuriya, H., Lee, E. S., Koonchanok, N. M., Geahlen, R. L., Ashendei, C.
L., et al. (1993). Kinase inhibitors from Polygonum cuspidatum. Journal of Natural
Products, 56, 1805–1810.
Johnson, T. O., Ermolieff, J., & Jirousek, M. R. (2002). Protein tyrosine phosphatase 1B
inhibitors for diabetes. Nature Reviews Drug Discovery, 1(9), 696–709.
Kenawi, I. M., Barsoum, B. N., & Youssef, M. A. (2005). Drug–drug interaction
between diclofenac, cetirizine and ranitidine. Journal of Pharmaceutical and
Biomedical, 37(4), 655–661.
Koren, S., & Fantus, I. G. (2007). Inhibition of the protein tyrosine phosphatase
PTPIB: Potential therapy for obesity, insulin resistance and type-2 diabetes
mellitus. Best Practice and Research Clinical Endocrinology and Metabolism, 21(4),
621–640.
the decease of activity in vitro. Compound 3, with different config-
uration of glucuronic acid moiety compared with 4, was inactive,
implying that the sugar configuration played a key role in the
inhibitory activity in vitro. Compound 10, with part molecular
structure of inactive compounds 6–9, showed strong inhibitory
activities, suggesting that the free carboxyl group in caffeic acid
was vital for the inhibitory activity, while the quinic acid group
did not contribute to the activity. Both compounds 1 and 11 belong
to naphthoquinone derivatives, revealed strong inhibitory activi-
ties. The PTP1B inhibitory activity of the compounds from C. paliu-
rus may contribute to the hypoglycaemic effect of this plant and
this study, therefore, partly explains the hypoglycaemic mecha-
nism of the plant.
Kraut-Cohen, J., Muller, W. J., & Elson, A. P. (2008). Protein–tyrosine phosphatase
epsilon regulates Shc signaling in
a kinase-specific manner – Increasing
coherence in tyrosine phosphatase signaling. Journal of Biological Chemistry,
283(8), 4612–4621.
Li, J., Lu, Y. Y., Su, X. J., Li, F., She, Z. G., He, X. C., et al. (2008). A nor-sesquiterpene
lactone and a benzoic acid derivative from the leaves of Cyclocarya paliurus and
their glucosidase and glycogen phosphorylase inhibiting activities. Planta
Medica, 74(3), 287–289.
Liu, L., Li, W., Koike, K., & Nikaido, T. (2004). Two new naphthalenyl glucosides and a
new phenylbutyric acid glucoside from the fruit of Juglans mandshurica.
Heterocycles, 6(63), 1429–1436.
Liu, L. Y., Sun, Y., Lauraa, T., Liang, X. F., Ye, H., & Zeng, X. X. (2009). Determination of
polyphenolic content and antioxidant activity of kudingcha made from Ilex
kudingcha C.J. Tseng. Food Chemistry, 112(1), 35–41.
Liverio, L., Puglisi, P. P., Morazzoni, P., & Bombardelli, E. (1994). Antimutagenic
activity of procyanidins from Vitis vinifera. Journal for the Study of Medicinal
Plants, 65, 203–209.
Loke, W. M., Proudfoot, J. M., Mckinley, A. J., Needs, P. W., Kroon, P. A., Hodgson, J.
M., et al. (2008). Quercetin and its in vivo metabolites inhibit neutrophil-
mediated low-density lipoprotein oxidation. Journal of Agricultural and Food
Chemistry, 56(10), 3609–3615.
Meng, L., Lozano, Y., Bombarda, I., Gaydou, E. M., & Li, B. (2008). Polyphenol
extraction from eight Perilla frutescens cultivars. Comptes Rendus Chimie, 1–10.
Miyase, T., Saitoh, H., Shiokawa, K., & Ueno, A. (1995). Six new presenegenin
glycosides, reiniosides A–F, from Polygala reinii root. Chemical and
Pharmaceutical Bulletin, 43, 466–472.
Nakanishi, T., Inatomi, Y., Murata, H., Ishida, S. S., Fujino, Y., Miura, K., et al. (2007).
Triterpenes and flavonol glucuronides from Oenothera cheiranthifolia. Chemical
and Pharmaceutical Bulletin, 55(2), 334–336.
Peterson, J., & Dwyer, J. (1998). Flavonoids: Dietary occurrence and biochemical
activity. Nutrition Research, 18, 1995–2018.
4. Conclusions
Water-soluble extract of C. paliurus exhibited strong inhibitory
activity against PTP1B (IC50 = 1.27 lg/mL), so it could be used as
a potential source for hypoglycaemic and anti-obesity functional
food. In the extract, both the naphthoquinone derivatives and phe-
nol–acidic compounds can be considered as promising classes of
PTP1B inhibitors. Undoubtedly, this study will provide fundamen-
tal knowledge for research and development of C. paliurus leaves.
In addition, further investigation and optimisation of these deriva-
tives might enable the preparation of new PTP1B inhibitors as a
functional food ingredient in the prevention and treatment of dia-
betes and obesity.
Acknowledgements
The authors are grateful to the financial support from the Na-
tional Natural Science Foundation of China (20872179 and
30472141), Science and Technology Commission of Shanghai
Municipality (STCSM) (08DZ1971504), Shanghai-SK Development
Fund (2004005-t) and the Innovation Fund for Graduate Student
of Shanghai Jiao Tong University.
Sanbongi, C., Takano, H., Osakabe, N., Sasa, N., Natsume, M., Yanagisawa, R., et al.
(2004). Rosmarinic acid in perilla extract inhibits allergic inflammation induced
by mite allergen, in
971–977.
a mouse model. Clinical and Experimental Allergy, 34,
Ueda, H., Yamazaki, C., & Yamazaki, M. (2002). Luteolin as an anti-inflammatory and
anti-allergic constituent of Perilla frutescens. Biological and Pharmaceutical
Bulletin, 25, 1197–1202.
Wang, H. H., Liu, B., Liu, X. Q., Zhang, J. W., & Xian, M. (2008). Synthesis of biobased
epoxy and curing agents using rosin and the study of cure reactions. Green
Chemistry, 10(11), 1190–1196.
Zabolotny, J. M., Bence-Hanulec, K. K., Stricker-Krongrad, A., Haj, F., Wang, Y.,
Minokoshi, Y., et al. (2002). PTP1B regulates leptin signal transduction in vivo.
Developmental Cell, 2(4), 489–495.
Zhang, W., Hong, D., Zhou, Y. Y., Zhang, Y. N., Shen, Q., Li, J. Y., et al. (2006). Ursolic
acid and its derivative inhibit protein tyrosine phosphatase 1B, enhancing
insulin receptor phosphorylation and stimulating glucose uptake. Biochimica et
Biophysica Acta, 1760, 1505–1512.
References
Agerstrand, M., Ruden, C.,
&
Wester, M. (2008). The Swedish environmental
An empirical
information and classification scheme for pharmaceuticals
–
investigation of the motivations, intentions and expectations underlying its
development and implementation. Toxicology Letters, 180, S177–S178.
Agrawal, P. K. (1992). NMR spectroscopy in the structural elucidation of
oligosaccharides and glycosides. Phytochemistry, 31, 3307–3330.
Ahmad, F., Azevedo, J. L., Cortright, R., Dohm, J. L.,
& Goldstein, B. J. (1997).
Alterations in skeletal muscle protein–tyrosine phosphatase activity and
expression in insulin-resistant human obesity and diabetes. Journal of Clinical
Investigation, 100, 449–458.
Zhou, S. H., Fang, Z. X., Lü, Y., Chen, J. C., Liu, D. H., & Ye, X. Q. (2009). Phenolics and
antioxidant properties of bayberry (Myrica rubra Sieb. et Zucc.) pomace. Food
Chemistry, 112(2), 394–399.
Alonso, A., Sasin, J., Bottini, N., Friedberg, I., Friedberg, I., Osterman, A., et al. (2004).
Protein tyrosine phosphatases in the human genome. Cell, 117(6), 699–711.
Bailey, G. S., & Williams, D. E. (1993). Potential mechanisms for food related
carcinogens and anti-carcinogens. Food Technology, 47, 105–118.