Arabian Journal of Chemistry (2015) 8, 803811

King Saud University

Arabian Journal of Chemistry

www.ksu.edu.sa
www.sciencedirect.com

ORIGINAL ARTICLE

New glycosidic constituents from fruits of Lycium

chinense and their antioxidant activities
I.M. Chung a, M. Ali b, P. Nagella a, A. Ahmad
a
b

a,*

Department of Applied Life Science, Konkuk University, Seoul 143-701, South Korea
Faculty of Pharmacy, Hamdard University, New Delhi 110062, India

Received 18 November 2012; accepted 24 May 2013

Available online 4 June 2013

KEYWORDS
Lycium chinense;
Solanaceae;
Fruits;
New constituents;
Antioxidant activities

Abstract Potential biologically active new constituents labd-3b, 9b-diol-3a-D-glucopyranosyl(2a 1b)-a-D-glucopyranosyl-(2b 1c)-a-D-glucopyranosyl-(2c 1d)-a-D-arabinofuranosyl-2dp-hydroxybenzoate (1) and a-D-glucuronopyranosyl (2 10 )-a-D-glucuronopyranosyl (20 100 )-a00
000
D-glucopyranosyl-2 -n-octadec-9 -enoate (2) along with b-sitosterol-b-D-glucoside were isolated
from the fruits of Lycium chinense. Their chemical structures were elucidated using detailed spectroscopic studies. The structure assignments are based on two-dimensional (2D)-NMR techniques
including COSY, HSQC, HMBC and NOESY experiments. Compounds 1 and 2 were evaluated
for antioxidant activities with three assay protocols such as diphenylpicrylhydrazyl (DPPH) radical
scavenging activity, reducing power and the phosphomolybdenum activity, compound 2 showed
more potential as compared with 1.
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1. Introduction
Lycium chinense Miller fruits (Fructus Lycii) known as
Gou-Qi-Zi in Chinese have long history of application as a
valuable tonic and health food supplement for improving
vision and maintaining good health. It is reputed to have
properties like nourishing the blood, enriching the yin,
tonifying the kidney and the liver, and moistening the lungs
(Peng et al., 2005). Fruits of L. chinense (Solanaceae),
* Corresponding author. Tel.: +82 2 450 3730; fax: +82 2 446 7856.
E-mail address: ateeque97@gmail.com (A. Ahmad).
Peer review under responsibility of King Saud University.

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distributed in northeast Asia, especially China, Japan, Korea,

and Taiwan, have been widely used as a tonic in traditional medicine. Potentially hepatoprotective glycolipid constituents and
determination of betain in L. chinense fruits have been
reported (Jung et al., 2005; Shin et al., 1999). Antimicrobial
compounds have also been reported from L. chinense roots
(Lee et al., 2005). The plant is reported to possess antibacterial,
anticancer and antioxidant properties (Lee et al., 2005; Zhang
et al., 2011; Wang et al., 2010). Antihepatotoxic activity and
chemical constituents from L. chinense fruits have been
reported (Chin et al., 2003). Variation in fruit sugar
zcomposition of Lycium barbarum and L. chinense of different
regions and varieties was also reported (Zheng et al., 2010).
Evaluation of antioxidant and other activities of compounds
from L. barbarum and L. chinense has also been reported (Li
et al., 2007; Ming et al., 2009). Some compounds were reported
in recent reports from L. chinense fruits (Jung et al., 2012; Chung

1878-5352 2013 Production and hosting by Elsevier B.V. on behalf of King Saud University.
http://dx.doi.org/10.1016/j.arabjc.2013.05.020

804
et al., 2013). As part of our ongoing investigations on the biologically active compounds from L. chinense fruits, we report here
the isolation and identication of two new compounds (12) together with known compound from the methanolic extract of
fruits of L. chinense. New compounds 1 and 2 were evaluated
for antioxidant activities with three assay protocols such as
diphenylpicrylhydrazyl (DPPH) radical scavenging activity,
reducing power and the phosphomolybdenum activity. The approach developed has proved useful in the study of the active
constituents in traditional Chinese medicines like L. chinense.
2. Experimental
2.1. General information
All chemicals used were of analytical grade. Hexane, ethyl acetate, chloroform, methanol, ethanol, water, sulphuric acid and
vanillin were purchased from Daejung Chemicals and Metals
Co. Ltd, Korea. Pre-coated TLC plates (layer thickness
0.25 mm), silica gel for column chromatography (70230 mesh
ASTM) and LiChroprep RP-18 (4063 lm) were from Merck
(Darmstadt, Germany). Previously isolated authentic standard
of b-sitosterol-b-D-glucoside is available. Optical rotation was
measured on an AA-10 model polarimeter (Instruments Ltd,
Seoul, South Korea). Both 1H and 13C NMR spectra were obtained on a Bruker Avance 600 high resolution spectrometer
operating at 600 and 150 MHz, respectively. This NMR machine
was available at the Seoul National University (SNU), Seoul,
South Korea and all NMR spectra were recorded at SNU.
NMR spectra were obtained in deuterated methanol using tetramethylsilane (TMS) as an internal standard, with chemical shifts
expressed in ppm (d) and coupling constants (J) in Hz. FAB MS
data were recorded on a JMS-700 (Jeol, Japan) spectrometer
instrument which was available at SNU, Seoul, South Korea.
IR spectra were recorded on an Innity Gold FT-IR (Thermo
Mattson, USA) spectrophotometer, which was available at Korea Institute of Science and Technology, Seoul, South Korea. The
sugars were determined using high performance liquid chromatography (HPLC, Waters Milfords, MA, USA) with a universal
evaporative lights scattering detector, column Eurospher 100
NH2, detector differential refractometer R401, mobile phase acetonitrile:water (4:1), ow rate of 1.0 ml/min, ambient temperature and 2 Mpa pressure standard samples of sugars were
obtained from Merck (Germany). The sugar solutions injected
into the column calibration lines for each sugars were made,
which were later used for assessing the concentrations corresponding to the different peaks in the chromatograms.
2.2. Preparation of the extracts
The fruits of L. chinense (3.1 kg) were immersed in methanol
(8 L) for three days at room temperature and then the
supernatant was concentrated under vacuum to yield 230 g
of the extract, which was suspended in water and extracted
with hexane, ethyl acetate and n-butanol successively to
produce 20.0 g, 10.1 g and 40 g of the extracts respectively.
2.3. Isolation of the compounds from n-butanol extract
The entire butanol extract was subjected to normal phase
column chromatography over silica gel (600 g) to yield 24

I.M. Chung et al.

fractions (each of 500 mL) with the following eluants: fractions 12 with chloroform, fractions 34 with chloroform:methanol (9.8:0.2, V:V), fractions 56 with
chloroform:methanol (9:6:0.4, V:V), fractions 78 with chloroform:methanol (9.4:0.6, V:V), fractions 910 with chloroform:methanol (9.2:0.8, V:V), fractions 1112 with
chloroform:methanol (9:1, V:V), fractions 1314 with chloroform:methanol (8.8:1.2, V:V), fractions 1516 with chloroform:methanol (8.5:1.5, V:V), fractions 1718 with
chloroform:methanol (8:2, V:V), fractions 1920 with chloroform:methanol (8.5:2.5, V:V) and fractions 2124 with methanol. All fractions were examined by TLC. Fractions 14 were
not further separated due to the low amount of the substance.
Fractions 78 (0.9 g) were crystallized after purication by column chromatography, yielding b-sitosterol-b-D-glucoside
(70 mg) whose identity was conrmed through comparison
of TLC and spectroscopic data with those of an authentic
sample. Fractions 1314 (4.4 g) were re-chromatographed over
LiChroprep RP-18 (ODS silica gel; 4063 lm: 200 g; each
fraction 100 mL). The elution was sequentially performed with
methanol and water to yield 20 fractions with the following
eluants: fractions 14 with water:methanol (8:2, V:V), fractions 58 with water:methanol (6:4, V:V), fractions 912 with
water:methanol (4:6, V:V), fractions 1316 with water:methanol (2:8, V:V), fractions 1720 with methanol. Fractions 912
were rechromatographed over Lichroprep RP18 ODS (80 g,
each fraction of 50 mL). The elution was sequentially performed with methanol containing 80%, 60%, 40%, 20%,
10%, and 0% of water to yield two new compounds 1 and 2.
3. Spectral data
3.1. Labd-3b, 9b-diol-3a-D-glucopyranosyl-(2a 1b)-a-Dglucopyranosyl-(2b 1c)-a-D-glucopyranosyl-(2c 1d)-a-Darabinofuranosyl-2d-p-hydroxybenzoate (1)
Light yellow viscous; [a]D20 + 25.1 (c 0.13, MeOH); 1H NMR
(MeOD, 600 MHz) d: 1.79, 1.84 (m, 2H, H-1), 1.98, 2.03 (m,
2H, H-2), 3.70 (dd, J = 4.9, 9.6 Hz, 1H, H-3), 2.27 (dd,
J = 7.2, 7.8 Hz, 1H, H-5), 1.53, 1.58 (m, 2H, H-6), 1.56,
1.60 (m, 2H, H-7), 1.96 (m, 1H, H-8), 1.67 (t, J = 6.0 Hz,
2H, H-11), 1.33, 1.37 (m, 2H, H-12), 1.60 (m, 1H, H-13),
1.41, 1.43 (m, 2H, H-14), 0.90 (t, J = 6.6 Hz, 3H, H-15),
1.25 (br s, 3H, H-16), 1.29 (br s, 3H, H-17), 0.92 (d,
J = 7.2 Hz, 3H, H-18), 1.27 (br s, 3H, H-19), 0.95 (d,
J = 7.8, 3H, H-20), 5.33 (d, J = 4.8, 1H, H-1a), 3.88 (dd,
J = 4.8, 5.2 Hz, 1H, H-2a), 3.87 (m, 1H, H-3a), 3.83 (m, 1H,
H-4a), 4.15 (m, 1H, H-5a), 3.30 (br s, 2H, H-6a), 5.31 (d,
J = 5.0 Hz, 1H, H-1b), 3.73 (dd, J = 3.6, 5.5 Hz, 1H, H-2b),
3.81 (m, 1H, H-3b), 3.65 (m, 1H, H-4b), 3.82 (m, 1H, H-5b),
3.32 (br s, 2H, H-6b), 4.36 (d, J = 6.0 Hz, 1H, H-1c), 4.01
(dd, J = 4.2, 6.0 Hz, 1H, H-2c), 3.69 (m, 1H, H-3c), 3.56 (m,
1H, H-4c), 3.97 (m, 1H, H-5c), 3.28 (br s, 2H, H-6c), 4.63
(d, J = 5.9 Hz, 1H, H-1d), 4.12 (dd, J = 4.8, 5.9 Hz, 1H, H2d), 3.62 (m, 1H, H-3d), 4.31 (m, 1H, H-4d), 3.34 (br s, 2H,
H-5d), 7.62 (dd, J = 3.0, 7.2 Hz, 1H, H-20 ), 7.71 (dd,
J = 2.9, 8.5 Hz, 1H, H-30 ), 7.70 (dd, J = 2.9, 7.9 Hz, H-50 ),
7.60 (dd, J = 3.0, 7.9 Hz, 1H, H-60 ); 13C NMR (MeOD,
150 MHz) d: 30.2 (C-1), 26.2 (C-2), 79.0 (C-3), 56.5 (C-4),
40.3 (C-5), 21.4 (C-6), 30.6 (C-7), 31.7 (C-8), 78.8 (C-9), 35.2
(C-10), 30.9 (C-11), 30.7 (C-12), 33.2 (C-13), 20.3 (C-14),

New glycosidic constituents from fruits of Lycium chinense and their antioxidant activities
11.5 (C-15), 23.8 (C-16), 24.1 (C-17), 14.5 (C-18), 25.1 (C-19),
14.5 (C-20), 105.3 (C-1a), 84.8 (C-2a), 71.6 (C-3a), 64.5 (C-4a),
74.0 (C-5a), 60.6 (C-6a), 101.3 (C-1b), 83.6 (C-2b), 66.9 (C-3b),
64.6 (C-4b), 78.0 (C-5b), 61.6 (C-6b), 101.1 (C-1c), 82.7 (C-2c),
66.8 (C-3c), 64.8 (C-4c), 77.1 (C-5c), 62.9 (C-6c), 109.3 (C-1d),
87.5 (C-2d), 65.4 (C-3d), 89.4 (C-4d), 64.4 (C-5d), 133.7 (C-10 ),
132.5 (C-20 ), 124.9 (C-30 ), 150.0 (C-40 ), 123.4 (C-50 ), 130.1 (C60 ), 169.4 (C-70 ); IR (KBr) mmax: 3510, 3420, 3395, 2922,
2852, 1738, 1625, 1556, 1430, 1380, 1246, 1072, 1028 cm1;
FAB-MS (positive ion mode) m/z 1049 [M + H]+
(C50H81O23) (1.5), 366 (10.5), 269 (15.7), 366 (10.5), 269
(15.7), 253 (11.3), 137 (23.1), 121 (82.6); HR-FABMS (positive
mode) m/z 1049.5159 [M + H] (calculated for C50H81O23,
1049.5169); ESI Mass (positive mode) m/z 413 (M + H -3
glucose + 1furanose).
3.2. a-D-glucuronopyranosyl (2 1)-a-D-glucuronopyranosyl
(2 1)-a-D-glucopyranosyl -200 -n-octadec-9000 -enoate (2)
Dark yellow semi-solid; [a]D20 + 33.1 (c 0.23, MeOH); 1H
NMR (MeOD, 600 MHz) d: 4.49 (d, J = 6.6 Hz , 1H, H-1),
4.09 (dd, J = 6.1, 6.6 Hz, 1H, H-2), 3.71 (m, 1H, H-3), 3.64
(m, 1H, H-4), 3.96 (d, J = 7.2 Hz, 1H, H-5), 4.83 (d,
J = 6.8 Hz, H-10 ), 4.01 (dd, J = 5.8, 6.6 Hz, H-20 ), 3.69 (m,
1H, H-30 ), 3.57 (m, 1H, H-40 ), 3.75 (d, J = 6.6 Hz, 1H, H50 ), 5.01 (d, J = 6.9 Hz, 1H, H-100 ), 4.35 (dd, J = 6.6, 6.9 Hz,
1H, H-200 ), 3.66 (m, 1H, H-300 ), 3.49 (m, 1H, H-400 ), 3.78 (m,
1H, H-500 ), 3.27 (br s, 2H, H-600 ), 2.80 (d, J = 4.2 Hz, 2.77,
J = 4.1 Hz, 2H, H-2000 ), 2.42 (m, 2H, H-3000 ), 2.20 (m, 2H, H4000 ), 1.98 (m, 2H, H-5000 ), 1.96 (m, 4H, H-6000 & 7000 ), 2.62 (m,
2H, H-8000 ), 5.02 (m, 2H, H-9000 & 10000 ), 2.58 (m, 2H, H-11000 ),
2.30 (m, 2H, H-12000 ), 2.01 (m, 2H, H-13000 ), 1.96 (m, 2H, H14000 ), 1.49 (m, 2H, H-15000 ), 1.33 (m, 2H, H-16000 ), 1.25 (br s,
2H, H-17000 ), 0.89 (t, J = 7.2 Hz, H-18000 ); 13C NMR (MeOD,
150 MHz) d: 97.8 (C-1), 74.9 (C-2), 73.2 (C-3), 72.3 (C-4),
78.1 (C-5), 179.4 (C-6), 98.3 (C-10 ), 74.3 (C-20 ), 73.0 (C-30 ),
71.9 (C-40 ), 77.7 (C-50 ), 176.3 (C-60 ), 99.4 (C-100 ), 83.3 (C-200 ),
71.2 (C-300 ), 69.6 (C-400 ), 76.4 (C-500 ), 62.8 (C-600 ), 169.0 (C1000 ), 54.0 (C-2000 ), 39.1 (C-3000 ), 32.4 (C-4000 ), 32.0 (C-5000 ), 27.5
(C-6000 & C-7000 ), 41.5 (C-8000 ), 120.9 (C-9000 ), 116.9 (C-10000 ),
40.7 (C-11000 ), 27.5 (C-12000 ), 26.7 (C-13000 & C-14000 ), 27.0 (C15000 & C-16000 ), 22.6 (C-17000 ), 15.0 (C-18000 ); IR mmax: (KBr):
3410, 3375, 3265, 2930, 2843, 1737, 1709, 1628, 1477, 1395,
1334, 1081, 980, 895 cm1; FAB-MS (positive mode) m/z
(rel. int.) 797 [M + H]+ (C36H61O19 (2.1), 427 (11.3), 370
(6.8), 281 915.8), 265 (9.7), 193 (9.8); HRFAB MS (positive
mode) m/z 797.3798 [M + H]+ (calculated for C36H61O19,
797.3807); ESI Mass (positive mode) m/z 426 [M-2 glucoronic
acid]+ and 829 [M-H + H2O2]+.
3.3. Acid hydrolysis of compound 1
Compound 1 (10 mg) was reuxed with 2 mL of 1 mol/L
hydrochloric acid:dioxane (1:1, V:V) in a water bath for 4 h.
The reaction mixture was evaporated to dryness and partitioned with chloroform and water four times, and each extract
was concentrated. The chloroform extract contained the aglycone portion, while the water extract possessed glycone portion (co-chromatographed on TLC (CHCl3:CH3OH:H2O:
AcOH at 16:9:2:2) with authentic sample determined by
HPLC.

805

3.4. Acid hydrolysis of compound 2

A solution of compound 2 (10 mg in tetrahydrofuran) was
added to 0.5 mL 1 N HCl and stirred at 80 C for 4 h. After cooling, the reaction mixture was diluted with H2O and extracted
with EtOAc (3 ml 3), yielded oleic acid and the aqueous layer
was subjected to TLC (CHCl3:CH3OH:H2O:AcOH at 16:9:2:2)
together with an authentic sample of D-glucose and to HPLC.
4. Antioxidant activity
Three assay protocols such as diphenylpicrylhydrazyl (DPPH)
radical scavenging activity, reducing power and the
phosphomolybdenum activity were used for the evaluation of
antioxidant activity of compounds (12) as described below:
4.1. Free radical scavenging activity
The antioxidant activity of compounds 1 and 2 based on the
scavenging activity of the stable 1,1-diphenyl-2-picrylhydrazyl
(DPPH) free radical, was determined by the method described by Katerere and Eloff (2005). The method is based
on the reduction of methanolic DPPH solution in the presence of a hydrogen donating antioxidant, due to the formation
of the non-radical form DPPHH by the reaction (Blois,
1958). Different concentrations (1.0, 2.0, 3.0, 4.0, and 5.0 mg
of 1 and 50, 100, 150, 200, and 250 lg of 2) of the tested samples (0.2 ml; compounds and BHT) were taken in different test
tubes with 4 ml of a 0.006% MeOH solution of DPPH.
Water (0.2 ml) in place of the compound was used as control.
Absorbance at 517 nm was determined after 30 min of incubation at 37 C. Radical scavenging activity was expressed as the
inhibition percentage and was calculated using the following
formula, % Radical scavenging activity = [(A0  A1)/
A0] 100, where A0 is the absorbance of the control, and A1
is the absorbance of the compound/standard.
4.2. Assay of reductive potential
The reductive potential of the compound was determined
according to the method of Dorman and Hiltunen (2004).
The reaction mixture comprises varying concentrations of
the compounds (1.0, 2.0, 3.0, 4.0, and 5.0 mg of 1 and 200,
400, 600, 800 and 1000 lg/ml of 2) in 1 ml of distilled water,
phosphate buffer (2.5 ml, 0.2 M, pH 6.6) and potassium ferricyanide [K3Fe(CN)6] (2.5 ml, 1%). The mixture was incubated
at 50 C for 20 min. A portion (2.5 ml) of trichloroacetic acid
(10%) was added to the mixture, which was then centrifuged at
1000 rpm for 10 min. The upper layer of the solution (2.5 ml)
was mixed with distilled water (2.5 ml) and FeCl3 (0.5 ml,
0.1%) and the absorbance was measured at 700 nm in a spectrophotometer. BHT was used as standard. Increased absorbance of the reaction mixture indicated increased reductive
potential. All analyses were run in triplicate and averaged.
4.3. Evaluation of antioxidant capacity by phosphomolybdenum
method
The total antioxidant capacity of compounds 1 and 2 was evaluated by the method (Prieto et al., 1999). An aliquot of 0.1 ml

806

I.M. Chung et al.

of sample solution (100 lg/ml) was combined with 1 ml of reagent solution (0.6 M sulphuric acid, 28 mM sodium phosphate and 4 mM ammonium molybdate). The tubes were
capped and incubated in a boiling water bath at 95 C for
90 min. After the samples had cooled to room temperature,
the absorbance of the aqueous solution of each was measured
at 695 nm against blank. A typical blank solution contained
1 ml of reagent solution and the appropriate volume of the
same solvent used for the sample and it was incubated under
same conditions as rest of the sample. The results are expressed
as equivalents of a-tocopherol (mg/g of compound).
5. Results and discussion
Compound 1, was obtained as a light yellow viscous mass from
chloroformmethanol (8.8:1.2, V:V) eluants. Its IR spectrum
showed characteristic absorption bands for hydroxyl groups
(3510, 3420, 3395 cm1), ester function (1738 cm1), and aromatic ring (1625, 1556, 1028 cm1). The FAB mass and 13C

NMR spectral data led to an established molecular formula

ion peak at m/z 1048 consistent with the molecular formula
of a diterpenic tetraglycoside esteried with an aromatic acid,
C50H80O23. The ion peaks arising at m/z 121 [HOC6H4CO]+,
137 [HOC6H4COO]+, 253 [HOC6H4COC5H8O4]+, and 269
[HOC6H4COC5H8O5]+, indicated that the aromatic acid
was linked to a hexose sugar unit. The fragmentation pattern
of compound 1 is shown in Fig. 2.
The 1H NMR spectrum of 1 showed four one-proton double doublets at d 7.62 (J = 3.0, 7.2), 7.71 (J = 2.9, 8.5 Hz),
7.70 (J = 2.9, 7.9) and 7.60 (J = 3.0, 7.9 Hz) assigned correspondingly H-20 , H-30 , H-50 and H-6 suggesting AA0 BB0 system. Four one-proton doublets at d 5.33 (J = 4.8 Hz), 5.31
(J = 5.0 Hz), 4.36 (J = 6.0 Hz), and 4.63 (J = 6.0 Hz) and
4.63 (J = 5.9 Hz) were attributed to a-oriented anomeric H1a, H-1b, H-1c and H-1d protons, respectively. The other sugar protons appeared between d 4.15 and 3.28. A one proton
double doublet at d 3.70 with coupling interactions of 4.9,
9.6 Hz was accounted to oxygenated methine H-3a proton.

20
11
19

HO

O
OH

4a

OH
HO
4b

HO
HO
4c

HO

10

1a

3a
6b

13

15

HO
5

OH
HO

17 16

4'

HO
HO

3b
6c

O
O
HO 1c

OH
O
4d OH

OH

18

O
O
OH 1b

4''

2c

5d

O
OH
2

7'

3'
4'

1'

6'

3'

2'

6'

4'

HO
HO

6''
4''

HO

OH
O

5'

m/z 121

1
COOH
O OH
OH
HO
HOOC

O
O
OH 1'
2'

O
O
OH

m/z 193

OO
HO

5''m/z

370

HO

2''

m/z 281

OCO(CH2)7CH=CH(CH2)7CH3
9'''

1'''

10'''

m/z 265

O CO (CH 2)7CH=CH (CH 2)7 CH3

18'''

2
Figure 1

m/z 177

HO
HO

OO
HO 1''
3''

m/z 253

m/z 137

COOH
5
O OH
OH 1

3'

m/z 269

OH

HO
HOOC

O
O
HO 1''

O
O
OH 1'

1d
2'

OH 3'' 2''
O
O
OH

HO

2d

3d

OH

6a

14

12

Chemical structures of compounds 1 and 2.

Figure 2

Mass fragmentation pattern of compounds 1 and 2.

New glycosidic constituents from fruits of Lycium chinense and their antioxidant activities
Three broad signals at d 1.25, 1.29 and 1.27 and two doublets
at d 0.92 (J = 7.2 Hz) and 0.95 (J = 7.8 Hz), all integrated for
three protons each were ascribed to tertiary methyl Me-16,
Me-17 and Me-19 and secondary methyl Me-18 and Me-20
respectively. A three proton triplet at 0.90 (J = 6.6 Hz), was
due to primary methyl Me-15 proton. The remaining methylene and methine resonated from d 2.27 to 1.33.
The 13C NMR spectrum of 1 and its one-dimensional
modications (ATP) showed that the compound was a glycoside with four sugar residues. The 13C NMR spectrum displayed signals for aromatic carbons from d 150.03 to 123.40,
ester carbon at d 169.49 (C-70 ), anomeric carbon at d 105.37
(C-1a), 101.35 (C-1b), 101.16 (C-1c), and 109.36 (C-1d), the
other sugar carbons between d 89.42 and 60.68, oxygenated
methine carbon at d 79.08 (C-3), hydroxyl-substituted quaternary carbon at d 78.84 (C-9), and other labdane carbons from
d 56.55 to 11.55. The presence of two sugar signals in the
deshielded region at d 109.36 (C-1d), and 89.42 (C-4d)
suggested a-furanoarabinose moiety in the sugar chain. The
presence of C-2a at d 84.81, C-2b at d 83.65, C-2c at d 82.72
and C-2d at d 87.75 supported C21 linkages of the sugar units.
Analysis of the spinspin coupling constants of the anomeric
carbon atoms to anomeric protons established that the sugars
adopted the furanose form with an axial anomeric proton.
The 1H1H COSY spectrum of 1 showed correlations of H3 with H2-2, H3-16 and H-1a; H-2a with H-1a, H-3a and H-1b;
H-2c with H-1c, H-3c and H-1d; H-20 with H-30 and H-60 ; H-5
with Me-16 and H2-6; and Me-20 with H-13, Me-15 and

Figure 3

807

H2-14. The HMBC spectrum of 1 exhibited interactions of

C-3 with H2-2, H3-17 and H-1a; C-9 with H-8, H3-18 and
H3-19; C-2a with H-3a and H-1b; C-1d with H-2d and H-2c;
C-4d with H2-5d, H-3d and H-2d; and C-70 with H-20 , H-60
and H-2d. The HSQC experiment showed key-correlations between the proton H-3 at d 3.70 and C-3 at d 79.08; H-1a at d
5.33 with C-1a at d 105.37; H-1b at d 5.31 with C-1b at d
101.35; H-1c at d 4.36 with C-1c at d 101.16; H-1d at d 4.63
with C-1d at d 109.36 and aromatic protons with respective
carbon signals. The COSY and TCOSY spectra showed close
spin systems belonging to sugar protons and enabled a determination of the monosaccharide composition of the carbohydrate part of each glycoside. These spectra exhibited a series
of close spin systems for the protons of rings A and B of the
aglycone and enabled their partial assignment. The ROESY
spectrum showed the bonding sequence of residues in the
tetrasaccharide and the site of attachment of the sugars to
the aglycone. The ROESY spectrum of 1 contained the usual
correlation peaks for a furanose with axial anomeric protons
H-1a/H-2a, H-1a/H-3a, H-1a/H-5a, and H-1a/H-6a; in
additions to correlation peaks H-1a/H-2b, H-1b/H-1c; H-1c/
H-2c, H-1c/H-1d; H-1d/H-2c, H-1d/H-2d, H-1d/H-5d; and
H-2d/H-20 and H-20 and 60 . This enables the sequence of residues in the tetrasaccharide. The NOESY spectrum of 1 showed
correlations of H-3a with Me-16 and H-5a; Me-19 with Me-18,
H2-1 and H2-11.
Acid hydrolysis of 1 yielded glucose and arabinose as
sugars (co-TLC and HPLC comparable). On the basis of the

Antioxidant activity of compound 1 at different concentration levels as measured by DPPH radical scavenging activity.

808

I.M. Chung et al.

foregoing discussion the structure of 1 has been established as

labd-3b, 9b-diol-3a-D-glucopyranosyl-(2 1)-a-D-glucopyranosyl-(2 1)-a-D-glucopyranosyl-(2 1)-a-D-arabinofuranosyl-2d-p-hydroxybenzoate. This is a new diterpene
glycoside.
Compound 2 (Fig. 1), was obtained as dark yellow viscous
mass from chloroformmethanol (8.8:1.2; V:V) eluants. It gave
positive test for glycosides. Its IR spectrum showed characteristic absorption bands for hydroxyl groups (3410, 3375,
3265 cm1), ester group (1737 cm1), carboxylic function
(1709 cm1) and unsaturation (1628 cm1). On the basis of
FAB mass and 13C NMR spectra the molecular weight of 2
was determined at m/z 797 [M + H]+ corresponding to the
molecular formula of a triglycosidic fatty acid ester
C36H61O19. The ion peaks arising at 265 [CH3
(CH2)7CHCH (CH2)7 CO]+, 281 [CH3 (CH2)7CHCH
(CH2)7 COO]+ and 427 [C6H10O6oleate]+ suggested that
oleic acid moiety was attached to the C6-sugar unit. The ion
peaks arising at 193 [C5H8O5COOH]+, and 370 [C5H8O5COOHC5H7O4COOH]+ supported the presence of two glucoronosidic units in the molecule. The fragmentation pattern
of compound 2 is shown in Fig. 2.
The 1H NMR spectrum of 2 exhibited a two-proton multiplet at d 5.02 assigned to vinylic H-9000 and H-10000 protons
respectively. Three one-proton doublets at d 4.49

Figure 4

(J = 6.6 Hz), 4.83 (J = 6.8 Hz) and d 5.10 (J = 6.9 Hz) were
ascribed to a-oriented H-1, H-10 and H-100 anomeric protons
respectively. The other sugar protons appeared from d 4.35
to 3.27. The presence of H-2, H-20 and H-200 signals as double
doublets in the deshielded region at d 4.09 (J = 6.6, 6.1 Hz),
4.01 (J = 6.6, 5.8 Hz), 4.35 (J = 6.6, 6.9 Hz) respectively indicated (2 1) linkage of the sugar units and location of ester
function at C-200 . A three-proton triplet at d 0.89
(J = 7.2 Hz) was accounted to terminal primary C-18000 methyl
protons. The remaining methylene protons resonated between
d 2.80 and 1.25.
The 13C NMR spectrum showed important signals for carboxylic carbons at d 179.40 (C-6) and 176.35 (C-60 ), anomeric
carbons at d 97.83 (C-1), d 98.38 (C-10 ), d 99.45 (C-100 ), and
other sugar carbons from d 83.33 to 62.89. The presence of
C-200 in the deshielded region at d 83.33 supported the existence
of the function at this carbon. The signals for fatty acids chain
resonated for unsaturated carbons at d 120.93 (C-9000 ) and
116.80 (C-10000 ), methylene carbons from d 54.01 to 22.63
and methyl carbon at d 15.06 (C-18000 ).
The 1H1HCOSYspectrum of 2 showed interactions of H-10
with H-20 , H-30 and H-2; H-100 with H-200 and H-20 ; H-9000 with
H-8000 and H2-11000 ; and H2-600 with H-500 . The HMBC spectrum
of 2 exhibited correlations of C-6 with H-5 and H-4; C-10 with
H-2, H-20 , H-30 ; C-100 with H-20 and H-200 ; C-1000 with H-200 ; and

Antioxidant activity of compound 2 at different concentration levels as measured by DPPH radical scavenging activity.

New glycosidic constituents from fruits of Lycium chinense and their antioxidant activities
C-9000 with H-10000 , H2-8000 and H-11000 . The HSQC spectrum of 2
showed important correlations of anomeric H-1, H-10 and
H-100 with C-1, C-10 and C-100 ; H-2 at d 4.09 with C-2 at d
74.93; H-200 at d 4.35 with C-200 at d 83.33; and vinylic protons
at d 5.02 with C-9000 and C-10000 . The ROESY spectrum of 2
showed correlation signals for axial anomeric protons H-1
with pyranose protons H-2, H-3, H-5 and H-10 ; H-20 with H10 , H-30 , H-40 and H-100 ; and H-200 with H-100 , H-300 and H22000 . This enables the sequence of sugar units and site of attachment of the trisaccharide to the aglycone.
Acid hydrolysis of 2 yielded oleic acid, glucoronic acid and
glucose. On the basis of above evidences, the structure of 2 has
been elucidated as a-D-glucuronopyranosyl (2 1)-a-D-glucuronopyranosyl
(2 1)-a-D-glucopyranosyl-200 -n-octadec9000 -enoate. This is a new glycosidic ester.
6. Biological activity
6.1. Free radical scavenging activity
Figs. 3 and 4 show the concentration dependent antioxidant
activity of compounds 1 and 2 at different concentration levels
as measured by the DPPH-radical scavenging assay. Compounds 1 and 2 were able to reduce the stable radical DPPH
to the yellow-coloured diphenylpicrylhydrazine. The IC50 value of compounds 1 and 2 were 3.94 mg/ml and 53.31 lg/ml
respectively. The DPPH activity of BHT showed a higher degree of free radical-scavenging activity than that of the compound at very low concentration points. The DPPH activity
of BHT exhibited 92.04% at 50 lg/ml concentration with an

Figure 5

809

IC50 value of 27.16 lg/ml (data not shown). This is similar

to other studies wherein they have reported that only 0.3 mg/
ml tocopherol, 0.23 mg/ml BHT and 0.1 mg BHA exhibited
a free radical scavenging activity equivalent to 3.9 mg/ml of
red bean and 10 mg/ml of sesame coat extract (Chang et al.,
2002; Chung et al., 2002).
6.2. Reducing power
As shown in Figs. 5 and 6 reducing power of compounds 1 and
2 increased with increasing concentration from 1.0 to 5.0 mg/
ml for compound 1 and 200 to 1000 lg/ml for compound 2.
The activity of BHT was higher than the test samples at each
concentration points (data not shown). This is in accordance
with the observations of several other workers wherein the
reducing power of BHT and tocopherol (Chung et al., 2002)
and BHA (Oktay et al., 2003) was higher than the extracts.
In the present study, compounds 1 and 2 from the butanol
fraction of methanol extract of lycium fruits exhibited a good
reducing power.
6.3. Antioxidant capacity by phosphomolybdenum method
The antioxidant capacity of compounds 1 and 2 was measured
spectrophotometrically through phosphomolybdenum method, which is based on the reduction of Mo (IV) to Mo (V)
by the sample analyte and the subsequent formation of green
phosphate/Mo (V) compounds with a maximum absorption
at 695 nm. The antioxidant capacity of compounds 1 and 2
was found to be 115. 95 and 131.74 mg/g respectively.

Reducing power of compound 1 at different concentration levels.

810

I.M. Chung et al.

Figure 6

Reducing power of compound 2 at different concentration levels.

7. Conclusion
The new compounds 1 and 2 were isolated from the butanol
fraction of methanolic extract of L. chinense fruits along with
known compound. Compounds 1 and 2 were evaluated for
antioxidant activities with three assay protocols as radical
scavenging activity, reducing power and phosphomolybdenum
activity, compound 2 showed more potential as a natural
antioxidant as compared with 1. The approach developed
has proved useful in the study of the active constituents in
traditional Chinese medicines.

Acknowledgements
Financial support for this research was provided by the Korea
Institute of Planning & Evaluation for Technology (IPET)
[111047-05-2-SB020] in Food, agriculture, Forestry and Fisheries of the Republic of Korea.

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