Flavonoid glycosides from the seeds of litchi chinensis

Article abstract of DOI:10.1021/jf104387y

Seven flavonoid glycosides, including one new (1) and five previously uncharacterized (3-7), were obtained from the seeds of lychee (Litchi chinensis Sonn. cv. Heiye) by means of repetitive column chromatography and high-performance liquid chromatography (HPLC) preparation. They were identified as litchioside D (1), (-)-pinocembrin 7-O-neohesperidoside (2), (-)-pinocembrin 7-O-rutinoside (3), taxifolin 4′-O-β-d-glucopyranoside (4), kaempferol 7-O-neohesperidoside (5), tamarixetin 3-O-rutinoside (6), and phlorizin (7) on the basis of spectroscopic analysis and comparison of their data to the values reported in the literatures. Among them, compounds 1, 4, and 5 showed in vitro antitumor activity against A549, LAC, Hep-G2, and HeLa cell lines in the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay.

Full text of DOI:10.1021/jf104387y

ARTICLE
pubs.acs.org/JAFC
Flavonoid Glycosides from the Seeds of Litchi chinensis
†
†
,‡
,†
†
†
Xinya Xu, Haihui Xie,* Jing Hao, Yueming Jiang, and Xiaoyi Wei
†
Key Laboratory of Plant Resources Conservation and Sustainable Utilization, South China Botanical Garden,
Chinese Academy of Sciences, Guangzhou 510650, People’s Republic of China
‡
Graduate University of Chinese Academy of Sciences, Beijing 100049, People’s Republic of China
ABSTRACT: Seven flavonoid glycosides, including one new (1) and five previously uncharacterized (3-7), were obtained from
the seeds of lychee (Litchi chinensis Sonn. cv. Heiye) by means of repetitive column chromatography and high-performance liquid
chromatography (HPLC) preparation. They were identified as litchioside D (1), (-)-pinocembrin 7-O-neohesperidoside (2),
0
(
3
-)-pinocembrin 7-O-rutinoside (3), taxifolin 4 -O-β-D-glucopyranoside (4), kaempferol 7-O-neohesperidoside (5), tamarixetin
-O-rutinoside (6), and phlorizin (7) on the basis of spectroscopic analysis and comparison of their data to the values reported in the
literatures. Among them, compounds 1, 4, and 5 showed in vitro antitumor activity against A549, LAC, Hep-G2, and HeLa cell lines
in the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay.
KEYWORDS: Litchi chinensis, lychee seed, flavonoid glycoside, litchioside D, cytotoxic activity
’
INTRODUCTION
(100-200 mesh, Qingdao Haiyang Chemical Co., Ltd., Qingdao,
China), Develosil ODS (S-75 μm, Nomura Chemical Co., Ltd., Seto,
Japan), macroporous resin Diaion HP-20 (Mitsubishi Chemical
Corporation, Tokyo, Japan), and Sephadex LH-20 (Amersham Bio-
sciences, Uppsala, Sweden). High-performance liquid chromatography
Lychee (Litchi chinensis Sonn.), the only member of the genus
Litchi in the family Sapindaceae, is a subtropical tree native to
China and now cultivated in many parts of the world. Its fruits are
popular in domestic and oversea markets because of their edible
and delicious arils and attractive appearance. The annual output
(
HPLC) was carried out on a Shimadzu LC-6AD liquid chromatograph
equipped with a Shimadzu RID-10A refractive index detector
Shimadzu Corp., Kyoto, Japan). YMC-Pack ODS-A columns (S-5
9
of lychee fruits in China exceeds 1.3 ꢀ 10 kg. As a byproduct of
(
the fruits, lychee seeds are mainly discarded as waste, although
they are used as a traditional Chinese medicine for the treatment
μm, 250 ꢀ 4.6 mm and 250 ꢀ 20 mm inner diameter, YMC Co.,
Ltd., Kyoto, Japan) were used for analytical and preparative purposes,
respectively. Thin-layer chromatography (TLC) was conducted on
precoated silica gel HSGF254 plates (Yantai Jiangyou Silica Gel Devel-
opment Co., Ltd., Yantai, China) and reversed-phase (RP)-18 F254S
plates (Merck Japan Ltd., Tokyo, Japan), and spot detection was
performed under fluorescent light (λ = 254 and 365 nm) and then
spraying 10% H SO in EtOH, followed by heating. 3-(4,5-Dimethyl-
1,2
of epigasteric pain and testicular swelling and pain. Pharmacolo-
gical studies revealed that lychee seeds possessed antihyperglycemic,
antihyperlipidemic, antiplatelet aggregation, antitumor, anti-
3
-5
viral, and antioxidant effects.
However, reports concerning
6,7
their biologically active components remain very scarce. To
clarify the structures and bioactivities of uncharacterized compo-
nents, we carried out further chemical investigation on the seeds.
In previous papers, we reported four eudesmane sesquiterpene
glucosides and their cytotoxic activity and seven A-type proantho-
cyanidins and two related compounds and their antioxidant and
antiviral activities. As a continuation of this work, we describe the
isolation and identification of seven flavonoid glycosides and their
in vitro antitumor activity in the present paper.
2
4
thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and naringinase
were purchased from Sigma Chemical Co. (St. Louis, MO). Roswell
Park Memorial Institute (RPMI)-1640 medium and admycin were
purchased from Gibco BRL (Gaithersburg, MD) and Shenzhen Main
Luck Pharmaceutical, Inc. (Shenzhen, China), respectively. Fetal calf
serum was obtained from Guangzhou Jinan Biomedicine Research and
Development Center (Guangzhou, China).
8
9
Plant Material. Lychee (L. chinensis cv. Heiye) fruits at commercial
maturity were collected from an orchard in Luogang District, Guangzhou,
China, in July 2007. A voucher specimen was deposited at the Herbarium
of South China Botanical Garden, Chinese Academy of Sciences, Guangz-
hou, China. The seeds were manually separated and ground to pieces with
a Santronic multifood processor.
’
MATERIALS AND METHODS
General Methods. Optical rotations were measured on a Perkin-
Elmer 343 polarimeter (Perkin-Elmer, Inc., Waltham, MA). Nuclear
magnetic resonance (NMR) spectra were recorded on a Bruker DRX-
Extraction and Isolation. Fresh lychee seed pieces (6,150 g)
were extracted 3 times with 95% EtOH (10 L ꢀ 3) at room temperature
4
00 NMR spectrometer (Bruker Biospin Gmbh, Rheistetten, Germany)
with the solvent residual peaks of dimethyl sulfoxide (DMSO)-d at δ
.50 and δ 39.52 as references. High-resolution electrospray ionization
6
H
(
25-32 °C) for 4 days each time. The concentration of the solution
2
C
under vacuum gave a reddish solid (934.86 g), of which 906.34 g was
mass spectrometry (HRESIMS) was obtained on a Bruker Bio TOF
IIIQ mass spectrometer (Bruker Daltonics, Inc., Billerica, MA), and
electrospray ionization mass spectrometry (ESIMS) data were acquired
on a MDS SCIEX API 2000 liquid chromatography/tandem mass
spectrometry (LC/MS/MS) instrument (Applied Biosystems, Inc.,
Forster, CA). Column chromatography was performed over silica gel
Received: November 13, 2010
Accepted: January 7, 2011
Revised:
January 6, 2011
Published: February 2, 2011
r 2011 American Chemical Society
1205
dx.doi.org/10.1021/jf104387y
|J. Agric. Food Chem. 2011, 59, 1205–1209

Journal of Agricultural and Food Chemistry
ARTICLE
1
3
dissolved in 0.8 L of water and then successively partitioned with
petroleum ether (0.8 L ꢀ 5), ethyl acetate (EtOAc, 0.8 L ꢀ 5), and
n-butanol (n-BuOH, 0.8 L ꢀ 5) to afford petroleum-ether-soluble
Table 1. C NMR Data for Compounds 1-3 and (-)-
Pinocembrin 7-O-β-D-Glucopyranoside (1a) in DMSO-d6
1
1
C
1
1a
2
3
(
38.80 g), EtOAc-soluble (91.65 g), and n-BuOH-soluble (151.60 g)
fractions after condensation to dryness in vacuo. The petroleum-ether-
soluble fraction (38.80 g) was subjected to silica gel (822 g) column
2
3
4
5
6
7
8
9
1
1
2
3
4
5
6
1
2
3
4
5
6
1
2
3
4
5
6
1
2
3
4
5
6
78.52
42.05
196.82
162.55
96.48
164.90
95.21
163.07
103.47
138.47
126.82
128.64
128.64
128.64
126.82
97.60
79.76
76.54
69.33
75.50
65.94
100.73
68.60
69.70
71.65
66.39
16.54
100.65
70.29
70.72
72.09
68.37
17.85
78.67
42.20
78.64
42.18
79.07
42.29
(
72 mm inner diameter ꢀ 490 mm) chromatography eluted with
CHCl /MeOH [10:0, 9.5:0.5, 9:1, 8.5:1.5, 8:2, and 7:3 (vol/vol) at
.0, 11.0, 12.0, 17.5, 10.0, and 7.5 L, respectively] to gain fractions
P1-P11 after pooling according to their TLC profiles. Fraction P8 (2.10
g), obtained from the elution of CHCl /MeOH of 8.5:1.5, was passed
through a Develosil ODS (4.2 g) precolumn, and the 80% MeOH/H
vol/vol) eluate (0.29 g) was separated by Develosil ODS (5.8 g)
column (12 mm inner diameter ꢀ 105 mm) chromatography eluted
with MeOH/H O [1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, and 8:2 (vol/vol) at
0 mL each] to gain fractions P8-1-P8-8. Fraction P8-4 (28 mg) was
196.89
162.58
96.66
196.83
162.92
96.53
196.46
162.40
96.62
3
9
165.37
95.54
165.06
95.21
165.02
95.38
3
2
O
(
162.98
103.31
138.50
126.80
128.64
128.72
128.64
126.80
99.58
162.63
103.39
138.48
126.74
128.63
128.70
128.63
126.74
97.77
162.35
103.39
138.39
126.61
128.44
128.47
128.44
126.61
99.41
0
0
2
6
0
0
0
0
0
submitted to Sephadex LH-20 column chromatography eluted with
MeOH to yield compound 7 (10.7 mg, 0.000 18%). The n-BuOH-
soluble fraction (141.20 g) was dissolved in water and passed through a
HP-20 (2800 g) column sequentially eluted with H O and MeOH. The
2
MeOH eluate (80.80 g) was subjected to silica gel (1600 g) column
0
0
(
92 mm inner diameter ꢀ 590 mm) chromatography eluted with
CHCl /MeOH [9:1, 8.5:1.5, 8:2, 7:3, and 6:4 (vol/vol) at 14 L each]
00
00
3
73.04
79.92
72.94
to attain fractions B1-B6. Fraction B3 (2.23 g), obtained from the
elution of CHCl /MeOH of 8.5:1.5 and 8:2, was separated by Develosil
3
76.33
77.06
76.26
0
0
0
0
0
0
0
69.48
69.20
69.62
ODS (45.0 g) column (18 mm inner diameter ꢀ 360 mm) chromatog-
0
77.11
76.60
75.56
raphy eluted with MeOH/H O [1:9, 2:8, 3:7, 4:6. 5:5, 6:4, and 7:3 (vol/
2
0
60.57
60.43
65.99
vol) at 450 mL each] to give fractions B3-1-B3-35. Fractions B3-21 and
00
00
00
00
(2 -Rha)
100.73
68.62
B3-22 (178.9 mg) were purified by HPLC using 49% MeOH/H
v/v) as the mobile phase at the flow rate of 5 mL/min to offer
compound 2 (t at 59.8 min, 8.2 mg, 0.000 15%), compound 3 (t at
4.1 min, 8.6 mg, 0.000 15%), and compound 6 (t at 41.4 min, 21.6 mg,
.000 39%). Fraction B5 (1.43 g), obtained from the elution of CHCl3/
2
O
00
(
(2 -Rha)
00
R
R
(2 -Rha)
69.71
5
0
R
000
000
00
(2 -Rha)
71.65
00
(2 -Rha)
66.38
MeOH of 8:2 and 7:3, was separated by Develosil ODS (28.8 g) column
18 mm inner diameter ꢀ 230 mm) chromatography eluted with
MeOH/H O [1:9, 2:8, 3:7, 4:6. 5:5, and 6:4 (vol/vol) at 300 mL each]
0
0
0
0
0
0
00
00
(2 -Rha)
16.52
(
000
000
000
000
000
00
(6 -Rha)
100.52
70.19
70.67
72.05
68.20
17.68
2
00
(6 -Rha)
to afford fractions B5-1-B5-30. Fractions B5-8 and B5-9 (76.9 mg)
were separated by Sephadex LH-20 column chromatography eluted with
MeOH to yield compound 4 (39.0 mg, 0.000 70%). Fractions B5-
00
(6 -Rha)
00
(6 -Rha)
00
1
8-B5-21 (348.2 mg) were separated by Sephadex LH-20 column
chromatography eluted with MeOH to supply compound 5 (7.1 mg,
.000 13%) and a residue, which was purified by HPLC using 48%
(6 -Rha)
0000
00
(6 -Rha)
0
MeOH/H O (vol/vol) as the mobile phase at the flow rate of 5 mL/min
to provide compound 1 (t at 51.9 min, 15.3 mg, 0.000 28%).
(1H, dd, J = 2.8, 12.5 Hz, H-2), 3.20 (1H, dd, J = 12.5, 17.2 Hz, H-3a), 2.84
(1H, dd, J = 2.8, 17.2 Hz, H-3b), 12.02 (1H, br s, OH-5), 6.10 (1H, d, J = 2.0
2
R
2
0
0
Litchioside D (1). Yellowish powder. [R]
D
- 95.3 (c 0.60,
, 400 MHz) δ: 5.66 (1H, dd, J = 2.8,
2.1 Hz, H-2), 3.17 (1H, dd, J = 12.1, 16.9 Hz, H-3a), 2.84 (1H, dd, J =
Hz, H-6), 6.18 (1H, d, J = 2.0 Hz, H-8), 7.53 (2H, dd, J = 1.6, 7.5 Hz, H-2 ,
1
0
0
0
0
MeOH). H NMR (DMSO-d
6
H-6 ), 7.44 (2H, dd, J = 7.5, 7.5 Hz, H-3 , H-5 ), 7.39 (1H, m, H-4 ), 5.15
00 000
(1H, d, J=7.7Hz, H-1 ), 5.07(1H, d,J=3.6Hz, H-1 ), 1.03(3H, d, J=6.5
1
2
0
00 13
.8, 16.9 Hz, H-3b), 12.00 (1H, br s, OH-5), 6.12 (1H, d, J = 1.8 Hz,
H-6), 6.14 (1H, d, J = 1.8 Hz, H-8), 7.54 (2H, dd, J = 1.8, 7.6 Hz, H-2 ,
Hz, H-6 ). C NMR (DMSO-d
6
, 100 MHz): see Table 1. ESIMS (þ)
0
þ þ
m/z: 257 [M - glucosyl - rhamnosyl þ H] , 587 [M þ Na] ; (-) m/z:
0
0
0
0
- - -
H-6 ), 7.44 (2H, dd, J = 7.6, 7.6 Hz, H-3 , H-5 ), 7.38 (1H, m, H-4 ), 5.17
255 [M - glucosyl - rhamnosyl - H] , 563 [M - H] , 599 [M þ Cl] .
(-)-Pinocembrin 7-O-Rutinoside (3). Yellowish powder.
00 00
(
1H, d, J = 7.6 Hz, H-1 ), 3.44 (1H, m, H-2 ), 3.52 (1H, dd, J = 8.9, 9.1,
0
0
00
00
00
000
20
1
H-3 ), 3.20 (1H, m, H-4 ), 3.62 (1H, m, H-5 ), 3.44 (1H, m, H-6 a),
.79 (1H, dd, J = 3.8, 10.2 Hz, H-6 b), 5.07 (1H, d, J = 2.8 Hz, H-1 ),
.58 (1H, m, H-2 ), 3.47 (1H, d, J = 3.0 Hz, H-3 ), 3.44 (1H, m,
H-4 ), 3.89 (1H, q, J = 6.4 Hz, H-5 ), 1.03 (3H, d, J = 6.5 Hz, H-6 ),
.52 (1H, br s, H-1 ), 3.65 (1H, m, H-2 ), 3.44 (1H, m, H-3 ), 3.15
1H, dd, J = 3.2, 8.4 Hz, H-4 ), 3.40 (1H, m, H-5 ), 1.08 (3H, d,
J = 5.7 Hz, H-6 ). C NMR (DMSO-d , 100 MHz): see Table 1. ESIMS
D 6
[R] - 31.4 (c 0.53, MeOH). H NMR (DMSO-d , 400 MHz) δ:
0
0
3
3
5.66 (1H, dd, J = 3.0, 12.2 Hz, H-2), 3.22 (1H, dd, J = 12.2, 17.2 Hz,
H-3a), 2.85 (1H, dd, J = 3.0, 17.2 Hz, H-3b), 11.96 (1H, br s, OH-5),
0
00
000
0
00
000
000
0 0
6.14 (2H, br s, H-6, H-8), 7.54 (2H, dd, J = 1.6, 7.7 Hz, H-2 , H-6 ), 7.44
0
000
0000
0000
0 0 0
(2H, dd, J = 7.7, 7.7 Hz, H-3 , H-5 ), 7.39 (1H, m, H-4 ), 4.98 (1H, d, J =
4
(
0
000
0000
00 0000 0000
7.4 Hz, H-1 ), 4.52 (1H, br s, H-1 ), 1.08 (3H, d, J = 6.2 Hz, H-6 ).
0
000 13
13
C NMR (DMSO-d
, 100 MHz): see Table 1. ESIMS (þ) m/z: 587
6
þ
6
þ
þ
-
(
þ) m/z:711[Mþ H] ,733[Mþ Na] ;(-) m/z: 255 [M - glucosyl -
[M þ Na] ; (-) m/z: 255 [M - glucosyl - rhamnosyl - H] , 563
-
-
-
-
-
rhamnosyl - rhamnosyl - H] , 709 [M - H] , 745 [M þ Cl] .
[M - H] , 599 [M þ Cl] .
-
-
0
HRESIMS (-) m/z: 745.2134 [M þ Cl] (calculated for C33
H
42ClO17
,
Taxifolin 4 -O-β-D-Glucopyranoside (4). Yellowish powder.
2
0
1
7
45.2116).
D 6
[R] - 20.4 (c 0.58, MeOH). H NMR (DMSO-d , 400 MHz) δ:
(
-)-Pinocembrin7-O-Neohesperidoside (2). Yellowish powder.
5.82 (1H, dd, J = 11.2 Hz, H-2), 5.04 (1H, d, J = 11.2 Hz, H-3), 11.89
(1H, br s, OH-5), 5.86 (1H, d, J = 1.8 Hz, H-6), 8.71 (1H, br s, OH-7),
20
1
[
D 6
R] - 34.0 (c 0.05, MeOH). H NMR (DMSO-d , 400 MHz) δ: 5.66
1
206
dx.doi.org/10.1021/jf104387y |J. Agric. Food Chem. 2011, 59, 1205–1209

Journal of Agricultural and Food Chemistry
ARTICLE
0
5
.90 (1H, d, J = 1.8 Hz, H-8), 6.96 (1H, d, J = 1.8 Hz, H-2 ), 7.12 (1H, d,
supernatant was carefully removed by pipet, and then 200 μL of DMSO
solution was added to each well and shaken for 15 min to dissolve
formazan crystals. The absorbance was measured on a Bio-Rad model
550 microplate reader (Bio-Rad Laboratories, Hercules, CA) at the
wavelength of 570 nm. MTT solution with DMSO (without both cells
and medium) was used as a blank control, and admycin was used as a
reference control. The cytotoxic activity of test compounds to prolifera-
tion of A549, LAC, Hep-G2, and Hela cells was calculated by SPSS 16.0
analytic software according to the following formula: cytotoxic activity
(%) = (A570 of control cells - A570 of treated cells)/A570 of control
cells ꢀ 100%. The 50% inhibitory concentration (IC ) was defined as
0
0
J = 8.4 Hz, H-5 ), 6.87 (1H, dd, J = 1.8, 8.4 Hz, H-6 ), 4.70 (1H, d, J = 7.1
, 100 MHz) δ: 82.68 (C-2), 71.51 (C-
), 197.67 (C-4), 163.35 (C-5), 96.11 (C-6), 166.92 (C-7), 95.08 (C-8),
0
0
13
Hz, H-1 ). C NMR (DMSO-d
6
3
1
3
0
0
62.48 (C-9), 100.48 (C-10), 131.83 (C-1 ), 115.64 (C-2 ), 146.55 (C-
0
0
0
0
00
), 145.61 (C-4 ), 116.36 (C-5 ), 119.33 (C-6 ), 102.24 (C-1 ), 73.34
00 00 00 00 00
(
C-2 ), 77.24 (C-3 ), 69.86 (C-4 ), 75.93 (C-5 ), 60.80 (C-6 ).
þ
þ
ESIMS (þ) m/z: 288 [M - glucosyl þ H] , 489 [M þ Na] ; (-)
-
-
m/z: 465 [M - H] , 501 [M þ Cl] .
Kaempferol 7-O-Neohesperidoside (5). Yellowish powder.
2
0
1
[
R]
D
- 57.2 (c 0.14, MeOH). H NMR (DMSO-d
6
, 400 MHz) δ:
5
0
1
(
(
2.50 (1H, br s, OH-5), 6.37 (1H, br s, H-6), 6.79 (1H, br s, H-8), 8.06
the concentration required to reduce the viability of untreated cell
cultures by 50%.
0 0 0 0
2H, d, J = 8.6 Hz, H-2 , H-6 ), 6.94 (2H, d, J = 8.6 Hz, H-3 , H-5 ), 5.24
00 000
1H, d, J = 7.6 Hz, H-1 ), 5.09 (1H, d, J = 3.3 Hz, H-1 ), 1.02 (3H, d, J =
0
00 13
6
1
9
1
1
6
.3 Hz, H-6 ). C NMR (DMSO-d
6
, 100 MHz) δ: 147.65 (C-2),
36.11 (C-3), 176.14 (C-4), 160.42 (C-5), 98.63 (C-6), 162.47 (C-7),
4.16 (C-8), 155.82 (C-9), 104.82 (C-10), 121.55 (C-1 ), 129.70 (C-2 ),
15.56 (C-3 ), 159.46 (C-4 ), 115.56 (C-5 ), 129.70 (C-6 ), 98.13 (C-
), 80.21 (C-2 ), 77.17 (C-3 ), 69.28 (C-4 ), 76.67 (C-5 ), 60.51 (C-
), 100.89 (C-1 ), 68.69 (C-2 ), 69.74 (C-3 ), 71.67 (C-4 ), 66.48
’ RESULTS AND DISCUSSION
The EtOH extract of lychee seeds was dissolved in water and
then sequentially partitioned with petroleum ether, EtOAc, and
n-BuOH. The resulting petroleum-ether- and n-BuOH-soluble
fractions were separated by repetitive column chromatography
over silica gel, Develosil ODS, and Sephadex LH-20 and pre-
parative HPLC to yield seven flavonoid glycosides (1-7).
Compound 1 was obtained as a yellowish powder with
negative optical rotation. Its molecular formula was deduced to
be C H O from quasi-molecular ion peaks at m/z 711 [M þ
0
0
0
0
0
0
0
0
0
0
00
00
00
00
000
000
000
000
0
00
000
þ
(
C-5 ), 16.52 (C-6 ). ESIMS (þ) m/z: 595 [M þ H] , 617 [M þ
þ
-
Na] ; (-) m/z: 285 [M - glucosyl - rhamnosyl - H] , 593 [M -
-
-
H] , 629 [M þ Cl] .
2
0
Tamarixetin 3-O-Rutinoside (6). Yellowish powder. [R]
5.3 (c 0.48, MeOH). H NMR (DMSO-d , 400 MHz) δ: 12.59 (1H, br
s, OH-5), 6.22 (1H, d, J = 1.5 Hz, H-6), 10.97 (1H, br s, OH-7), 6.43
1H, d, J = 1.5 Hz, H-8), 7.50 (1H, d, J = 1.8 Hz, H-2 ), 7.03 (1H, d, J =
.5 Hz, H-5 ), 7.71 (1H, dd, J = 1.8, 8.5 Hz, H-6 ), 3.85 (3H, s, OCH
.37 (1H, d, J = 7.0 Hz, H-1 ), 4.40 (1H, br s, H-1 ), 0.98 (3H, d, J =
6
, 100 MHz) δ: 156.42 (C-2),
33.64 (C-3), 177.46 (C-4), 161.23 (C-5), 98.81 (C-6), 164.30 (C-7),
D
-
1
3
3 42 17
6
6
þ
-
H] and 709 [M - H] in the ESIMS spectrum in combination
with its NMR data as well as a negative-ion peak at 745.2134
[M þ Cl] in the HRESIMS spectrum. The H NMR spectrum
showed two meta-coupling doublets at δ 6.12 and 6.14 (1H each,
J = 1.8 Hz, H-6, H-8)], five aromatic protons at δ 7.38 (1H, m,
0
(
-
1
0
0
8
5
6
1
9
1
3
),
0
0
0000
0
000 13
.0 Hz, H-6 ). C NMR (DMSO-d
0
0
0
H-4 ), 7.44 (2H, dd, J = 7.6, 7.6 Hz, H-3 , H-5 ), and 7.54 (2H,
0
0
0
0
3.71 (C-8), 156.51 (C-9), 104.04 (C-10), 121.53 (C-1 ), 111.42 (C-2 ),
dd, J = 1.8, 7.6 Hz, H-2 , H-6 ), and three aliphatic doublets at
δ 5.66 (1H, J = 2.8, 12.1 Hz, H-2), 3.17 (1H, J = 12.1, 16.9 Hz,
H-3a), and 2.84 (1H, J = 2.8, 16.9 Hz, H-3b), suggesting the
0
0
0
0
50.10 (C-3 ), 145.91 (C-4 ), 115.69 (C-5 ), 122.52 (C-6 ), 55.66
00 00 00 00
(OCH
3
), 101.31 (C-1 ), 74.10 (C-2 ), 76.44 (C-3 ), 69.81 (C-4 ),
11
0
0
00
0000
0000
7
3
6
5.80 (C-5 ), 66.86 (C-6 ), 100.81 (C-1 ), 70.40 (C-2 ), 70.65 (C-
presence of a pinocembrin moiety. Moreover, the spectrum
0
000
0000
0000
þ
0000
), 71.86 (C-4 ), 68.29 (C-5 ), 17.77 (C-6 ). ESIMS (þ) m/z:
exhibited three anomeric protons at δ 5.17 (1H, d, J = 7.6 Hz,
þ
00 000
H-1 ), 5.07 (1H, d, J = 2.8 Hz, H-1 ), and 4.52 (1H, br s,
25 [M þ H] , 647 [M þ Na] ; (-) m/z: 315 [M - glucosyl -
-
-
-
0000 000
H-1 ) and two methyls at δ 1.03 (3H, d, J = 6.5 Hz, H-6 ) and
rhamnosyl - H] , 623 [M - H] , 659 [M þ Cl] .
0000
1.08 (3H, d, J = 5.7 Hz, H-6 ), suggesting a β-glucosyl and two
13
Enzymtic Hydrolysis of Compound 1. A solution of com-
pound 1 (2.8 mg) and naringinase from Penicillium decumbens (Product
N1385, 390 IU/g, 2 mg) in 1 mL of acetate buffer (pH 4.4) was stirred
at 38 °C for 4 h. The reaction solution was extracted with ethyl ether
R-rhamnosyl moieties. As shown in Table 1, the C NMR
spectrum presented signals of a carbonyl at δ 196.82 (C-2), 12
aromatic carbons at δ 95.21 (C-8), 96.48 (C-6), 103.47 (C-10),
0
0
(
1 mL ꢀ 3) after neutralization with 0.1 M NaOH. The concentration of
the combined ethyl ether solution yielded a residue, which was separated
by silica gel column chromatography eluted with CHCl /MeOH (9:1,
and 126.82-164.90, 3 anomeric carbons at δ 100.73 (C-1 ),
000
0000
1
00.65 (C-1 ), and 97.60 (C-1 ), 14 oxygenated carbons
ranging from δ 65.94 to 79.76, an aliphatic methylene at δ
2.05 (C-3), and 2 methyls at δ 17.85 and 16.54. These data
3
10
vol/vol) to afford compound 1a (1.0 mg), which was identified as (-)-
pinocembrin by a comparison of its ESIMS and H NMR data and
- 46.8 (c 0.08, CHCl ) to those reported in the literature.
D 3
Cytotoxic Assay. Human lung cancer A549, human pulmonary
carcinoma LAC, human hepatoma Hep-G2, and human cervical carci-
noma HeLa cell lines were generously provided by Guangzhou Jinan
Biomedicine Research and Development Center, Guangzhou, China.
The cells were maintained in RPMI-1640 medium plus 10% heat-
inactivated fetal bovine serum in a humidified atmosphere with 5%
4
1
indicated that compound 1 was a pinocembrin triglycoside. This
was supported by enzymatic hydrolysis of compound 1 with
naringinase, which yielded (-)-pinocembrin. In comparison to
20
11
[
R]
11
the data of (-)-pinocembrin 7-O-β-D-glucopyranoside, carbon
resonances for the pinocembrin skeleton in compound 1
were consistent with the literature data, whereas values of
0
0
00
C-1 -C-6 for the glucosyl moiety were shifted by -1.98,
þ6.72, þ0.21, -0.15, -1.61, and þ5.37 ppm, respectively
CO
2
at 37 °C. The cytotoxic activity of compounds 1-7 was determined
using the MTT colorimetric assay as previously described by
Mosmann. Briefly, cancer cells were seeded into wells of a 96-well
(
Table 1). These findings suggested that the glucosyl moiety
12
was linked to C-7 of the pinocembrin skeleton and two rhamosyl
00 00
moieties were connected to C-2 and C-6 of the gulcosyl
13
4
flat-bottom microtiter plate at 5 ꢀ 10 cell/mL in 195 μL of culture
moiety. In the heteronuclear multiple-bond correlation
medium. A serial 2-fold dilution of test compounds was made in DMSO.
Each serial solution of 5 μL was added to a well, including 195 μL of cell
culture medium. The final concentrations of each test compound were
00
(
HMBC) spectrum, long-range correlations from H-1 (δH
000 00
.17) to C-7 (δ 164.90), from H-1 (δ 5.07) to C-2 (δ
C H
5
C
0
000
00
6
.25, 12.5, 25, 50, and 100 μM. The plate was incubated at 37 °C in a
humidified atmosphere with 5% CO . After 72 h, 10 μL of 5 mg/mL
MTT solution was added to each well and incubated for 4 h. The
79.76), and from H-1 (δ
H
4.52) to C-6 (δ
C
65.94) were
observed, confirming the aforementioned connections. Further-
more, the proton and carbon values of three sugar moieties in
2
1
207
dx.doi.org/10.1021/jf104387y |J. Agric. Food Chem. 2011, 59, 1205–1209

Journal of Agricultural and Food Chemistry
ARTICLE
Figure 1. Structures of compounds 1-7 from the seeds of L. chinensis.
Table 2. Cytotoxic Activity of Compounds 1-7 (IC , μM)
activity against all four cell lines, with IC50 values ranging from
5
0
1.82 to 17.58 μM. However, the other flavonoid glycosides (2, 3, 6,
compounds
A549
LAC
Hep-G2
HeLa
and 7) were inactive (IC50 > 100 μM), except that compound 2
showed a weak activity against the HeLa cell line. It is noted that
the moderate activity of compound 2 against P-388 lymphocytic
leukemia, with an IC value of 2.58 μg/mL (= 4.57 μM), was
1
57.38
0.79
>100
>100
11.58
7.93
>100
>100
20.48
0.030
>100
>100
4.22
23.98
37.64
2
>100
>100
17.50
0.53
>100
>100
15.15
50
3
>100
1.82
0.051
>100
>100
22.64
22
previously reported, while the activity of compounds 1, 4, and 5
against A549, LAC, Hep-G2, and HeLa cancer cells is reported for
the first time. The discovery of the cytotoxic compounds 1, 4, and 5
suggested that they might also be involved in the antitumor activity of
lychee seeds.
4
5
0.020
>100
>100
79.50
6
7
admycin
It is worth noting that the isolated known compounds were
found to have other biological activities according to literature
reports. (-)-Pinocembrin 7-O-neohesperidoside (synonym:
onychin, 2) showed biological activities of inhibiting the pro-
0
0
00
compound 1 were consistent with those of apigenin 7-O-(2 ,6 -
13
di-R-L-rhamnopyranosyl)-β-D-glucopyranoside. Accordingly,
0
0
compound 1 was determined to be (-)-pinocembrin 7-O-(2 ,
liferation of vascular smooth muscle cells and preventing H O -
2 2
23,24
0
0
6
-di-R-L-rhamnopyranosyl)-β-D-glucopyranoside (Figure 1), which
induced apoptosis in ECV304 endothelial cells.
Tamarixetin
was a new flavonone glycoside and trivially named litchioside D.
The isolated known compounds were identified as (-)-
3-O-rutinoside (6) demonstrated inhibitory activity against
osteoclast differentiation by 40 and 63% at the concentrations
14,15
25
pinocembrin 7-O-neohesperidoside (2),
(-)-pinocembrin
of 0.1 and 1.0 μg/mL. The dihydrochalcone phlorizin (7)
16
0
17
7
-O-rutinoside (3), taxifolin 4 -O-β-D-glucopyranoside (4),
was a natural product and dietary constituent found in a
number of fruit trees, and its principal pharmacological action
was to produce renal glucosuria and block intestinal glucose
absorption through inhibition of the sodium-glucose sym-
porters located in the proximal renal tubule and mucosa of the
18
kaempferol 7-O-neohesperidoside (5), tamarixetin 3-O-rutino-
1
9
20,21
side (6), and phlorizin (7)
by interpretation of their
1
13
spectroscopic data ( H and C NMR and ESIMS) and by com-
parison to reported data. Among them, compounds 3-7 were
isolated from this species for the first time.
2
6
small intestine.
Cytotoxic Activity. The in vitro cytotoxic activity of seven
isolated flavonoid glycosides was evaluated against human lung
cancer A549, human pulmonary carcinoma LAC, human hepa-
toma Hep-G2, and human cervical carcinoma HeLa cell lines
using the MTT colorimetric assay. Their IC50 values on the
viability of aforementioned cancer cell lines after 72 h of
incubation were presented in Table 2. Kaempferol 7-O-neohe-
speridoside (5) exhibited significant cytotoxic activity against all
of the test cell lines, with IC50 values of 0.53, 7.93, 0.020, and
In conclusion, seven flavonoid glycosides were isolated in
purity from the seeds of lychee (L. chinensis var. Heiye). Their
structures were identified by the spectroscopic method, including
NMR and HRESIMS. Litchioside D (1) was a new flavonone
triglycoside. The known ones, except (-)-pinocembrin 7-O-
neohesperidoside (2), were obtained from this species for the
first time. Among these flavonoid glycosides, kaempferol 7-O-
neohesperidoside (5) possessed more potent in vitro cytotoxic
activity against A549, LAC, Hep-G2, and HeLa cancer cells than
12
0
0.051 μM, respectively, which were more potent than the reference
admycin and litchioside D (1) and taxifolin 4 -O-β-D-glucopyr-
compound admycin. Litchioside D (1) also showed potent activity
anoside (4) also showed the activity toward the test cell lines,
suggesting that they could be contributors to the antitumor
activity of lychee seeds.
against LAC and Hep-G2 cells (IC50 = 0.79 and 0.030 μM).
0
Taxifolin 4 -O-β-D-glucopyranoside (4) demonstrated moderate
1
208
dx.doi.org/10.1021/jf104387y |J. Agric. Food Chem. 2011, 59, 1205–1209

Journal of Agricultural and Food Chemistry
ARTICLE
’
AUTHOR INFORMATION
(20) Qin, X. D.; Liu, J. K. A new sweet dihydrochalcone-glucoside
from leaves of Litocarpus pachyphyllus (Kurz) Rehd. (Fagaceae). Z.
Naturforsch., C: J. Biosci. 2003, 58, 759–761.
(21) Nie, R. L.; Takashi, T.; Zhou, J.; Tanaka, O. Phlorizin and
trilobatin, sweet dihydrochalcone-glucosides from leaves of Lithocarpus
litseifolius (Hance) Rehd. (Fagaceae). Agric. Biol. Chem. 1982, 46, 1933–
Corresponding Author
*Telephone/Fax: þ86-20-37252537. E-mail: xiehaih@scbg.ac.cn.
Funding Sources
This work was supported by the Knowledge Innovation Program
of the Chinese Academy of Sciences (KSCX2-YW-R-218 and
KSCX2-EW-J-28) and a SCBG Fund for Talented Young
Scholars (SCBG200720).
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dx.doi.org/10.1021/jf104387y |J. Agric. Food Chem. 2011, 59, 1205–1209