International Immunopharmacology 7 (2007) 162 166

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Immunomodulatory and anticancer activities of flavonoids extracted

from litchi (Litchi chinensis Sonn.) pericarp
Mouming Zhao a,, Bao Yang a,, Jinshui Wang a , Yang Liu a ,
Limei Yu a , Yueming Jiang b
a
College of Light Industry and Food Science, South China University of Technology, Guangzhou 510640, People's Republic of China
b
South China Botanical Garden, Chinese Academy of Sciences, Guangzhou 510650, People's Republic of China
Received 22 July 2006; received in revised form 21 August 2006; accepted 8 September 2006

Abstract

The litchi pericarp extract was subjected to partition by hexane, ethyl acetate and water. Epicatechin, proanthocyanidin B2 and
proanthocyanidin B4 were isolated and purified from the ethyl acetate fraction by reverse-phase high performance liquid
chromatography. The immunomodulatory activities of epicatechin, proanthocyanidin B2, proanthocyanidin B4 and the ethyl
acetate fraction were examined using proliferation of mouse splenocytes. The results showed all these samples had much higher
stimulatory effects on splenocyte proliferation than that of the reference, rutin. Epicatechin and the ethyl acetate fraction showed a
significantly (P b 0.05) stimulatory effect when the concentration was up to 12.5 g/ml. Proanthocyanidin B2 and proanthocyanidin
B4 exhibited little lower stimulatory effects than epicatechin and the ethyl acetate fraction. The anti-breast cancer activities of
epicatechin, proanthocyanidin B2, proanthocyanidin B4 and the ethyl acetate fraction were also evaluated. Epicatechin and
proanthocyanidin B2 had lower cytotoxicities to human breast cancer cell MCF-7 and human embryolic lung fibroblast than
paclitaxel.
2006 Elsevier B.V. All rights reserved.

Keywords: Litchi pericarp; Epicatechin; Proanthocyanidin B4; Proanthocyanidin B2; Immunomodulatory activity; Anticancer activity

1. Introduction approximately 15% of the total weight of fresh fruit, and

Litchi (Litchi chinensis Sonn.) is an exotic fruit in
Southeast Asia, especially in China. As litchi fruit is
accepted gradually by consumers for its delicious taste
and attractive shape, the fruit is planted in other semi-
tropical regions [1,2]. Litchi pericarp tissues account for
comprise of significant amounts of flavonoids [3]. Thus,
it can be taken as an important source of flavonoids.
Flavonoids play some important pharmacological roles
against diseases, such as cardiovascular disease, cancer,
inflammation and allergy [4,5]. The role of the immune
system has become increasingly important in the under-
standing of the mechanisms that are involved in disease

prevention. Isoflavone daidzein could stimulate murine

The first two authors contributed equally to this work. nonspecific immunity, activate humoral immunity and
Corresponding authors. Zhao is to be contacted at Tel./fax: +86 20
87113914. Yang, Tel./fax: +86 20 37252960.
enhance cell-mediated immunity [6,7]. Epidemiological
E-mail addresses: femmzhao@scut.edu.cn (M. Zhao), studies have indicated the relationship between flavonoid
yangbao@china.com.cn (B. Yang). intake and reduced risk of certain cancers. In many studies
1567-5769/$ - see front matter 2006 Elsevier B.V. All rights reserved.
doi:10.1016/j.intimp.2006.09.003
 M. Zhao et al. / International Immunopharmacology 7 (2007) 162166
of dietary prevention of cancer, model of breast cancer has
been prominent in assessing the impact of a wide variety of
flavonoids for their efficacy in inhibiting cancer [8,9]. A
reduced risk of breast cancer incidence has been associated
with a high intake of genistein and moderate consumption
of red wine [10,11]. The suggested mechanisms for breast
cancer prevention effect of flavonoids are multifaceted,
including effects on signal transduction pathways involved
in cell proliferation, antioxidant activities, and modulations
of enzyme activity associated with estrogen biosynthesis
and metabolic pathways of carcinogens [12].
Wang et al. [13,14] reported the anticancer activity of
water-soluble ethanolic extract of litchi fruit pericarp
against human breast cancer and hepatocellular carcino-
ma in vitro and in vivo, but the compositions of the
water-soluble extract were not identified. Our previous
work indicated that the major flavonoids in ethyl acetate
fraction of litchi pericarp extract (LPE) were identified as
epicatechin, proanthocyanidin B2 and proanthocyanidin
B4 [15]. For a better exploration of the litchi fruit
pericarp tissues, it was necessary to understand the bio-
logical activities of these compounds and ethyl acetate
fraction. In the present study, immunomodulatory and
anti-breast cancer activities of epicatechin, proanthocya-
nidin B2, proanthocyanidin B4 and the ethyl acetate
fraction on mouse splenocytes and human breast cancer
cells MCF-7 were investigated, respectively.
2. Materials and methods
2.1. Plant materials
Fresh fruits of litchi (L. chinensis Sonn.) at the com-
mercially mature stage were picked from a commercial orchard
in Guangzhou, China. Fruits were selected for uniformity of
shape and colour prior to storage at 4 C in refrigerator.
2.2. Chemicals
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro-
mide (MTT), dimethyl sulfoxide (DMSO), -mercaptoethanol,
L-glutamine, paclitaxel and rutin were purchased from Sigma
Chemical Co (St Louis, MO, USA), while RPMI-1640 medium
was purchased from GIBCO/BRL Invitrogen (Caithershurg,
MD). Fetal calf serum (FCS), penicillin and streptomycin were
obtained from Zhongshan University (Guangzhou, China). All
other chemicals used were of analytical grade.
2.3. Animals
Normal female BALB/c mice (23 months old) from
Zhongshan University (Guangzhou, China) were housed under
standard environmental conditions and fed with standard
pellets and tap water.
163
2.4. Extraction of flavonoids
Pericarp tissues (5 g, dry weight) were extracted for 2 h at
30 C in 400 ml 85% ethanol by the methods of Lee and
Wicker, and Argolo et al. with a minor modification [16,17].
After filtering the extract through Whatman No. 1 paper, the
residue was re-extracted and filtered a second time. Filtrates
were combined and dried using a rotary evaporator at 40 C.
The dried extract was re-dissolved in 100 ml water and then
partitioned with 300 ml of hexane and ethyl acetate sequen-
tially. The ethyl acetate fraction was dried using a rotary
evaporator at 40 C, prior to further purification.
2.5. Separation and purification of flavonoids by reverse-
phase high performance liquid chromatography
The ethyl acetate fraction was loaded to a reverse-phase
high performance liquid chromatography (RPHPLC) proce-
dure on a polystyrene/divinyl column (100 6.4 mm i.d.)
(Pharmacia, Swiss) for isolation of epicatechin, proanthocya-
nidin B2 and B4. Elution conditions were as follows: solvent
A, water/formic acid (98:2, v/v); solvent B, acetonitrile/water/
formic acid (80:19:1, v/v/v); isocratic for 4 min with 3% B
following from 3 to 50% B within 14 min at a flow rate of 1 ml/
min. The RPHPLC profiles were recorded at 280 nm. The
RPHPLC can collect about 150 g for each compound at one
time. More than one hundred times were used repeatedly to
collect enough samples. The purity of these three compounds
was higher than 99%, by HPLC analysis with standards.
2.6. Cell line and culture
Human breast cancer MCF-7 and human embryonic lung
fibroblast (HELF) cell lines were obtained from immune system
analysis laboratory of Zhongshan University (Guangzhou,
China). The cells were maintained in RPMI-1640 complete
medium in a humidified 5% CO2 atmosphere at 37 C.
Mice were killed by cervical dislocation and then spleens
were removed aseptically. Single cells were prepared by
mincing spleen fragments and pressing through a stainless
200-mesh screen in RPMI complete medium [18]. An amount
of 1 107 cells was placed in a 16-mm well and incubated for
3 h in 5% CO2 at 37 C. The supernatant together with the non-
adherent cells was collected by centrifugation at 1000 rpm for
10 min. The cell pellets were re-suspended in RPMI complete
medium and then adjusted to 1 106 cells/ml [19].
RPMI-1640 complete medium was supplemented with
10% fetal calf serum (FCS), 2 mM L-glutamine, 50 M -
mercaptoethanol, penicillin (100 U/ml) and streptomycin
(100 g/ml).
2.7. In vitro proliferation assay
The proliferation stimulatory effect of various litchi
pericarp components on splenocytes and the cytotoxicity to
both MCF-7 and HELF cells were determined by the MTT
assay [20]. All cells were seeded into 4 wells of a 96-well flat-
164 M. Zhao et al. / International Immunopharmacology 7 (2007) 162166
bottom microtiter plate at 1 106 cell/ml in 100 l RPMI
complete medium. Various litchi pericarp components (0
500 g/ml), paclitaxel (0100 g/ml) and rutin (0200 g/ml)
were added to the medium, respectively, giving a final volume
of 200 l. The plate was incubated at 37 C in a humidified
atmosphere with 5% CO2. After 44 h, 50 l of MTT solution
(5 mg/ml) was added to each well and incubated for 4 h. The
supernatant was removed carefully by pipetting. DMSO
solution (100 l) was added to each well and shaken for
15 min. The absorbance at 570 nm was measured with a
microplate reader (Bio-Rad, Richmond, CA), using wells but
without cells as blanks. All experiments were performed in
triplicate. The cytotoxicity of various litchi pericarp compo-
nents to the proliferation of MCF-7 and HELF was calculated
as cytotoxicity (%) = (A570 of control cells A570 of treated
cells) / A570 of control cells 100%. The stimulatory effect of
various litchi pericarp components on the proliferation of
splenocytes was calculated as the percentage = (A570 of treated
cells A570 of control cells) / A570 of control cells 100%.
2.8. Statistical analyses
All the data were expressed as means standard deviation
(SD) of three replicated determinations. Statistical calculations
by OriginPro Version 7.5 software (OriginLab Corporation,
USA) were carried out. One way of variance analysis was
applied for determining differences between results of samples.
Values of P b 0.05 were considered as significantly different.
3. Results and discussion
3.1. Separation and purification of flavonoids
Ethyl acetate exhibited a good dissolving capacity to most
of flavonoids and can be used to classify flavonoids in plant
extract [21,22]. Our previous study indicated that the ethyl
acetate fraction of flavonoids accounted for 83.1% of the total
quantity in LPE. The RPHPLC profile showed that three major
flavonoids in the ethyl acetate fraction of LPE were identified
as epicatechin, proanthocyanidin B2 and proanthocyanidin B4
[15]. They were collected and used for evaluation of immuno-
modulatory and anti-breast cancer activity.
3.2. Immunomodulatory activity of flavonoids
Splenocyte proliferation from female BALB/c mice species
cultured in the presence of different samples was used to evaluate
cell stimulatory effects on the splenocyte growth. As shown in
Fig. 1, all samples used in this study had good stimulatory
effects. When the concentrations of epicatechin and the ethyl
acetate fraction were up to 12.5 g/ml, the lowest concentration
used in this study, significantly (P b 0.05) stimulatory effect on
splenocyte proliferation was observed. The concentration of
epicatechin, proanthocyanidin B2, proanthocyanidin B4 and the
ethyl acetate fraction to obtain 50% of stimulatory effect was 84,
99, 120 and 67 g/ml, respectively. There was a 110.5 5.2% of
stimulatory effect when 200 g/ml ethyl acetate fraction was
Fig. 1. The stimulatory effects of epicatechin, proanthocyanidin B2,
proanthocyanidin B4 and the ethyl acetate fraction of LPE on
proliferation of mouse splenocytes. A, epicatechin; B, proanthocya-
nidin B2; C, proanthocyanidin B4; D, rutin and E, the ethyl acetate
fraction. A dose-dependent stimulatory effect on the growth of mouse
splenocyte is observed. Results are means SD of triplicate indepen-
dent experiments.
used. Proanthocyanidin B2 and proanthocyanidin B4 had
significantly (P b 0.05) stimulatory effects when the concentra-
tion used was up to 25 g/ml, and exhibited little lower
stimulatory effects compared with epicatechin and the ethyl
acetate fraction. The reference, rutin, had a significantly
(P b 0.05) stimulatory effect when the concentration was up to
50 g/ml. Previous studies indicated that many plant extracts
could stimulate the proliferation of splenocyte [23,24]. Kong
et al. reported that propolis flavone could promote lymphocyte
proliferation [23]. In the present study, strong stimulatory effects
of these flavonoids on splenocyte proliferation suggested that the
litchi pericarp components could be used as an additional drug
constituents against some inflammations or other diseases.
3.3. Anti-breast cancer activity of flavonoids
Anti-breast cancer activities of various litchi pericarp
components were evaluated in terms of MCF-7 and HELF
proliferation. As shown in Fig. 2, paclitaxel, being approved
by FDA of USA as an anticancer drug, showed a strong
cytotoxicity to MCF-7 and HELF. When the concentration of
paclitaxel used in this study was up to 25 g/ml, the
cytotoxicity to MCF-7 was beyond 50%. The cytotoxicities
to MCF-7 and HELF were 72.5% and 62.4%, respectively,
when 100 g/ml paclitaxel was used. The results suggested
that paclitaxel had good inhibitory effect on cancer cell
proliferation, but it could destroy normal cell seriously. The
cytotoxicities of epicatechin and proanthocyanidin B2 to MCF-
7 were higher than that of proanthocyanidin B4 and the ethyl
acetate fraction. The IC50 to MCF-7 of epicatechin and
proanthocyanidin B2 was 102 and 99 g/ml, respectively.
Even when the concentration of proanthocyanidin B4 and the
ethyl acetate fraction was up to 500 g/ml, their cytotoxicities to
MCF-7 couldn't reach 50% yet. There was no obvious
improvement of cytotoxicity to MCF-7 when the concentration
of epicatechin or proanthocyanidin B2 increased from 125 to
500 g/ml. The IC50 to HELF of epicatechin, proanthocyanidin
 M. Zhao et al. / International Immunopharmacology 7 (2007) 162166
B2, proanthocyanidin B4 and the ethyl acetate fraction was 231,
254, 452 and 421 g/ml, respectively. Proanthocyanidin B4 and
ethyl acetate fraction showed a stronger cytotoxicity to HELF
than to MCF-7. When 62.5 g/ml was used, slightly stimulatory
165
effects were observed on MCF-7 proliferation. According to the
above results, proanthocyanidin B4 and the ethyl acetate fraction
could be unsuitable for cancer inhibition. Wang et al. [13]
reported anticancer activity of LPE against human breast cancer,
but they only used the water-soluble fraction of LPE for the
experiment in vivo and in vitro. In this study, the ethyl acetate
fraction and the major flavonoids of it were used to determine
their anticancer activities. Thus, it is better to exactly understand
the immunomodulatory activity and anti-breast cancer effect of
these compounds from the litchi pericarp.
A number of studies have suggested that flavonoids might
play a protective role in preventing breast cancer [25,26]. In
most reports, isoflavonoids have a significant effect on the
prevention of breast cancer. However, information about the
effect of flavanol on the breast cancer was relatively few.
Rodgers and Grant [27] suggested that the possible mechanisms
responsible for anti-breast cancer prevention by flavanol and
flavonol might involve in xenobiotic metabolising enzymes that
alter metabolic activation of potential carcinogens. Le Bail et al.
[28] mentioned that some of flavones and flavanones could alter
hormone production and inhibit aromatase to prevent the
development of breast cancer cells. The exact inhibitory
mechanisms of epicatechin, proanthocyanidin B2, proanthocya-
nidin B4 and the ethyl acetate fraction from litchi pericarp tissues
on the proliferation of breast cancer cells are needed to be
investigated.
4. Conclusion
Significant amounts of flavonoids existed in pericarp
tissues of litchi fruits. Three major flavonoids, epica-
techin, proanthocyanidin B2 and proanthocyanidin B4,
were obtained. By in vitro trials of mouse splenocyte
proliferation, significantly stimulatory effect was found
for these three flavonoids and the ethyl acetate fraction of
LPE. In terms of in vitro trials of MCF-7 and HELF
proliferation, proanthocyanidin B4 and ethyl acetate
fraction showed a stronger inhibitory effect on HELF
than MCF-7, while epicatechin and proanthocyanidin B2
had lower cytotoxicities to MCF-7 and HELF than
paclitaxel.
Acknowledgement
The financial support provided by National Natural
Science Foundation of China (No. 30425040), Guangz-
hou Scientific Research Foundation (No. 2005Z2-E0151)
Fig. 2. The cytotoxicities of epicatechin, proanthocyanidin B2,
proanthocyanidin B4 and the ethyl acetate fraction to proliferations
of MCF-7 and HELF. A, epicatechin; B, proanthocyanidin B2; C,
proanthocyanidin B4; D, the ethyl acetate fraction and E, paclitaxel.
Significant cytotoxicities of epicatechin and proanthocyanidin B2 to
the growth of MCF-7 and HELF are observed. Results are means SD
of triplicate independent experiments.
166 M. Zhao et al. / International Immunopharmacology 7 (2007) 162166
and Guangdong Provincial Agricultural Research Foun-
dation (No. 2003A203507) was greatly appreciated.
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