1. Phytomedicine 21 (2014) 647–655
Contents lists available at ScienceDirect
Phytomedicine
journal homepage: www.elsevier.de/phymed
Structurally characterized arabinogalactan from Anoectochilus
formosanus as an immuno-modulator against CT26 colon
cancer in BALB/c mice
Li-Chan Yanga
, Chang-Chi Hsiehb
, Ting-Jang Lua
, Wen-Chuan Linc,∗
a
Institute of Food Science and Technology, National Taiwan University, Taipei, Taiwan
b
Department of Animal Science and Biotechnology, Tunghai University, Taichung, Taiwan
c
School of Medicine, Graduate Institute of Basic Medical Science, China Medical University, Taichung, Taiwan
a r t i c l e i n f o
Article history:
Received 19 September 2013
Accepted 31 October 2013
Keywords:
Anoectochilus formosanus
Polysaccharide
Anti-cancer
Arabinogalactan
Immuno-modulator
Immunotherapy
a b s t r a c t
In this study, the innate immuno-modulatory effects and anti-cancer action of arabinogalactan (AG), a
derivative of a well-known orchid, Anoectochilus formosanus, were investigated. The innate immuno-
modulatory effects of AG were determined in vitro using RAW 264.7 cells for microarray analysis, and
in vivo using BALB/c mice administrated with AG at 5 and 15 mg/kg intra-peritoneally for 3 weeks. The
anti-cancer activity of AG was evaluated by CT26 colon cancer-bearing BALB/c mice. The microarray
analysis was performed to evaluate the innate immunity and demonstrated that AG significantly induced
the expression of cytokines, chemokines, and co-stimulatory receptors, such as IL-1␣, CXCL2, and CD69.
An intraperitoneal injection of AG in mice increased the spleen weight, but not the body weight. The
treatment of mitogen, LPS significantly stimulated splenocyte proliferation in AG treated groups. The
AG treatment also promoted splenocyte cytotoxicity against YAC-1 cells and increased the percentage
of CD3+
CD8+
cytotoxic T cells in innate immunity test. Our experiments revealed that AG significantly
decreased both tumour size and tumour weight. Besides, AG increased the percentage of DC, CD3+
CD8+
T cells, CD49b+
CD3−
NK cells among splenocytes, and cytotoxicity activity in tumour-bearing mice. In
addition, the immunohistochemistry of the tumour demonstrated that the AG treatments increased the
tumour-filtrating NK and cytotoxic T-cell. These results demonstrated that AG, a polysaccharide derived
from a plant source, has potent innate immuno-modulatory and anti-cancer activity. AG may therefore
be used for cancer immunotherapy.
© 2013 Elsevier GmbH. All rights reserved.
Introduction
Polysaccharides have been used as complementary and alter-
native medicines (CAMs) to treat tumours in Asian countries. It
was recently reported that almost 50% of people in the United
Stated with cancer use both conventional and CAM (Lu et al., 2011).
Plant-based sources and micro-organisms have been critical in the
development of cancer drugs, such as Taxol, Camptothecin, and
Krestin (Wani et al., 1971; Hsiang et al., 1985; Ren et al., 2012).
These commercial cancer drugs were originally derived from phy-
tomedicine. Recently, a Phase III clinical colorectal cancer trial of a
␤-glucan derived from yeast was performed in the United States
∗ Corresponding author at: School of Medicine, China Medical University, No. 91
Hsueh Shih Road, Taichung, Taiwan. Tel.: +886 4 22053366; fax: +886 4 22053764.
E-mail address: wclin@mail.cmu.edu.tw (W.-C. Lin).
(Biothera, 2013). Krestin, a polysaccharide derived from mush-
rooms, was approved as a prescription drug for the treatment
of gastric cancer in Japan (Sullivan et al., 2006). These polysac-
charides were presumably designed for immune potentiating and
immunotherapy (Lu et al., 2011).
Anoectochilus formosanus is mostly cultivated in Asia, and is
a popular traditional medicine for the treatment of cancer (Shyur
et al., 2004). This orchid species is not under international protec-
tion and conservation. The aqueous extract of A. formosanus has
been reported to generate anti-tumour and splenocyte prolifer-
ation activities in mice when administrated orally (Tseng et al.,
2006). In addition, extracts of A. formosanus have been reported to
induce apoptosis in human MCF-7 breast cancer cells (Shyur et al.,
2004). However, the anti-tumour mechanism and compounds of A.
formosanus remain unclear.
The objective of this study was to determine whether the puri-
fied arabinogalactan from A. formosanus could stimulate the innate
0944-7113/$ – see front matter © 2013 Elsevier GmbH. All rights reserved.
http://dx.doi.org/10.1016/j.phymed.2013.10.032

2. 648 L.-C. Yang et al. / Phytomedicine 21 (2014) 647–655
Fig. 1. High performance size exclusion chromatography (HPSEC) of AG. HPSEC was
carried out using a HPLC system equipped with RI detector and TSKgel guard col-
umn PWH (75 mm × 7.5 mm i.d.; TOSOH, Tokyo, Japan), TSKgel G4000PWXL, and
G2500PWXL (300 mm × 7.8 mm i.d., TOSOH) connected in series. The molecular
weight of pullulan standards was 708 kDa, 375 kDa, 200 kDa, 107 kDa, 47.1 kDa,
21.1 kDa, 9.6 kDa and 5.9 kDa. The standard curve axis is right off the figure.
immune response in mice to assess the anti-tumour activity of AG
in a CT26-bearing mouse model of colon cancer, and to identify the
anti-tumour immunity of AG.
Materials and methods
Arabinogalactan preparation
Purified arabinogalactan (AG), was isolated from A. formosanus
tissue cultured on the Yu-Jung farm (Puli, Taiwan). The plant tis-
sue cultivation technology of A. formosanus has become popular.
Previous research has described the preparation process and iden-
tified AG as a Type II arabinogalactan with ␤-(1 → 3, 1 → 6) galactan
backbone (Yang et al., 2012). AG was precipitated by adding ethanol
into aqueous extract of A. formosanus and then treated with a
total dietary fibre kit (Megazyme, Ireland) to obtain indigestible
polysaccharide. The indigestible polysaccharide was fractionated
by anion-exchange chromatography on a column of DEAE 650M,
and eluted with a 20 mM tris buffer, followed by a sodium chloride
gradient (0–0.3 M) to collect the AG. The A. formosanus AG used
in this study was pharmaceutical grade AG with a purity of >99%
according to phenol-sulphuric method, protein, and the nuclear
acid contamination in the AG was negligible (absorbance at 280
and 260 nm wavelengths was close to zero). The monosaccharide
composition of AG was arabinose, galactose, glucose, and mannose
in a ratio of 22.4:56.5:15.4:5.4 (Yang et al., 2012). High perfor-
mance size exclusion chromatography (HPSEC) was used to identify
the molecular weight (Mr.) and purity of fractional collected
AG. HPSEC using a TSKgel guard column PWH (75 mm × 7.5 mm
i.d.; TOSOH, Tokyo, Japan), TSKgel G4000PWXL, and G2500PWXL
(300 mm × 7.8 mm i.d., TOSOH) connected in series. The column
was eluted with 0.3 N sodium nitrate solution containing 0.02%
sodium azide with a flow rate of 0.8 ml/min at 65 ◦C. Peaks were
detected by an Interferometric Refractometer (Wyatt, CA, USA).
The average Mr. of the AG was estimated by comparison with the
retention time of pullulan standard P-82 kit (molecular weights
of standards: P-800, 708 kDa; P-400, 375 kDa; P-200, 200 kDa; P-
100, 107 kDa; P-50, 47.1 kDa; P-20, 21.1 kDa; P-10, 9.6 kDa; and P-5,
5.9 kDa; Shodex, Kanadawa, Japan). As shown in Fig. 1, the results of
HPSEC illustrated that the molecular weight distribution of AG was
a symmetrical peak with average Mr. in 29 kDa. The average Mr. of
AG used in this experiment was the same to previous report (Yang
et al., 2012). In addition, the symmetrical peak of HPAEC and carbo-
hydrate content (>99%) proved that AG was a purity polysaccharide,
not a polysaccharide mixture.
An endotoxin assay was performed using a ToxinSensorTM
chromogenic LAL endotoxin assay kit (GenScript, NJ, USA) to ensure
that the AG was not contaminated with lipopolysaccharide (LPS).
The endotoxin assay results showed that the endotoxin contents of
100 ␮g/ml AG were less than 0.01 EU/ml.
Cell culture and microarray analysis
Murine macrophages RAW 264.7, murine YAC-1 and murine
colon cancer cells CT26 were obtained from the Food Industry
Research and Development Institute (Hsinchu, Taiwan), and were
cultured in Dulbecco’s modified Eagle’s medium (DMEM, Invitro-
gen, USA), and supplemented with 10% (v/v) foetal bovine serum
(FBS, Gibco, USA), 100 ␮g/ml of streptomycin, and 100 U/ml peni-
cillin (Gibco, USA) at 37 ◦C in a humidified atmosphere containing
5% CO2.
RAW 264.7 cells were harvested 6 h after stimulation with AG
(at 100 ␮g/ml). Total RNA was isolated from the cells using a TRIzol
kit (Invitrogen, USA) according to the manufacturer’s instructions.
Equal amounts of total RNA were pooled from 3 independent
samples. The RNA quantity and purity were assessed using Nano-
Drop ND-1000. Pass criteria absorbance ratios were established as
A260/A280 ≥1.8, and A260/A230 ≥1.5 indicating acceptable RNA
purity. An Agilent RNA 6000 nano assay kit was used to deter-
mine the renewable identification number (RIN) values and RNA
integrity. The pass criteria for the RIN value were established at ≥6,
indicating acceptable RNA integrity. Gel electrophoresis was used
to evaluate gDNA contamination. The target was prepared using an
Eberwine-based amplification method with an Amino Allyl Mes-
sageAmp II aRNA Amplification Kit (Ambion, AM1753) to generate
amino-allyl antisense RNA (aa-aRNA). Labelled aRNA coupled with
NHS-CyDye was prepared and purified before hybridization. The
purified coupled aRNA was quantified using NanoDrop ND-1000.
Labelled aRNA was hybridized to the Mouse OneArray® MOA 2.0
GeneChip (OneArray, Hsinchu, Taiwan). All procedures followed
the Phalanx Hybridization Protocol. Normalized intensities were
obtained through median scaling performed on a data set with-
out flagged or control data. Statistical analysis was performed
on technical replicates to assess reproducibility based on the
Pearson correlation coefficient. Furthermore, data were analyzed
by performing a pair-wise ratio calculation that included con-
ducting probe filtering, normalization, pair-wise comparison, and
error-weighted modelling based on sample groups. A statistically
significant set of differentially expressed genes was established at
log 2 |Fold change| ≥ 1 and p < 0.05.
Animals
Eight-week old male BALB/c mice were obtained from the
National Laboratory Animal Centre (Taipei, Taiwan). The experi-
mental animals received adequate care and humane treatment, and
all the animal experimental protocols, as stipulated in the insti-
tutional guidelines of the China Medical University for the use of
laboratory animals, were followed. The animals were housed in an
air-conditioned room (21–24 ◦C) and received humane care under
12 h of light (8:00 a.m.–8:00 p.m.), and were allowed free access to
food pellets and water throughout the study.
Innate immuno-modulatory of AG in BALB/c mice
Twenty-four BALB/c male mice were intraperitoneally injected
on a daily basis with AG (5, 15 mg/kg) or saline for 3 weeks. Each
group contained 8 mice. The body weight of each animal was

3. L.-C. Yang et al. / Phytomedicine 21 (2014) 647–655 649
measured once a week until the final day of treatment. Finally, the
animals were euthanized using CO2. Their spleens were removed
and weighed under sterile conditions, and then used for the spleno-
cytes preparation.
Tumour suppression by AG in CT26 bearing mice
CT26 carcinoma cells (1 × 106 cells/mouse) were subcuta-
neously inoculated into the BALB/c mice on Day 0, and were
subsequently randomly separated into groups. From Day 2, the
mice received daily intraperitoneal injections with AG (5, 15 mg/kg)
or saline. After Day 2, the mice in the positive control group received
intraperitoneal injections with 25 mg/kg of 5-fluorouracil (5-FU,
Pharmachemie BV, Haarlem, Nederland) every second day. From
the onset of the anti-tumour experiment, the body weight of the
mice was measured every 3 days, and tumours were measured
every 3 days starting from Day 7. The tumour sizes were calcu-
lated based on the following formula: volume (cm3) = 0.5 × A × B2,
where A was the longest length and B was the shortest length of
the tumour (Tomayko and Reynolds, 1989). The mice were eutha-
nized using CO2 anaesthesia on Day 21. The spleens and tumours
were immediately removed and weighed. Tumours were placed in a
10% neutral formaldehyde buffer to attain immunohistochemistry
stains.
Flow cytometry analyses of splenocytes
Splenocytes were prepared from innate and tumour bearing
mice using the method described by Harding (2001). The spleens
were aseptically removed, and placed in cold Roswell Park Memo-
rial Institute (RPMI) 1640 medium (Gibco, NY, USA). The spleens
were then teased apart and passed through a nylon mesh (BD Fal-
con, CA, USA). The spleen cell suspensions were hemolyzed by 0.1
and 2 M HBSS to lyse the red blood cells, and 5 mL RPMI 1640
medium containing 10% FBS, 2 mM l-glutamine (Gibco), 100 ␮M 2-
2-mercaptoethanol (Gibco), 1.0 mM sodium pyruvate (Gibco), and
gentamycin (Gibco) was then added. The single spleen cell suspen-
sions were centrifuged at 4 ◦C, 300 × g for 10 min for collection. The
cell viability of splenocytes was counted using the FACSCaliburTM
flow cytometer (Becton-Dickinson, CA, USA) with a propidium
iodide (PI)/RNAase (Invitrogen) stain. All antibodies used for the
flow cytometry analyses were purchased from eBioscience (CA,
USA). The cells (5 × 105 cells/25 ␮l) from the splenocytes were
stained with 10 ␮l of FITC-labelled anti-mouse CD4, PE-labelled
CD8, PE-Cy5-labelled anti-mouse CD3, PE-Cy5 anti-mouse CD3e,
FITC-labelled anti-mouse CD49b, PE-labelled NK-T, FITC-labelled
anti-mouse CD14, PE-labelled CD11c, PE-Cy5-labelled CD137, FITC-
labelled CD86, PE-Cy5-labelled MHCII at 4 ◦C for 30 min in the
dark. After incubation, unlabelled antibodies were washed with
3 mL PBS containing 0.05% sodium azide and then re-suspended
in a 200 ␮l FACS buffer containing 2.0% FBS and 0.05% sodium
azide (Sigma–Aldrich, MO, USA). Cells were then analyzed with
FACscan and the data were analyzed with CellQuest software (BD
Biosciences). The results of flow cytometry were presented as per-
centages of positive fluorescent cells.
Measurements of splenocytes proliferation from AG treated mice
Splenocytes were isolated from the BALB/c mice and were
administrated with saline or AG (5, 15 mg/kg). The splenocytes
were seeded into a 96-well plate at 5 × 106 cells/ml in a 100 ␮l of
RPMI 1640 medium. Thereafter, the splenocytes were stimulated
with a Con A (5 ␮g/ml), LPS (10 ␮g/ml), or RPMI 1640 medium in a
final volume of 200 ␮l. After 48 h of incubation, the measurement
of the splenocytes proliferation was determined with MTS assay
(Wang et al., 2010), and the plate was subsequently read at 492 nm
using a TRAID LT ELISA reader. The cell viability percentage was
calculated using the following formula:
Cell viability percentage =
OD sample
OD control
× 100
Cytotoxic activity of splenocytes against YAC-1 cell
Splenocytes (Effector-E) were harvested from the mice in both
an innate immune experiment and an anti-tumour experiment.
The splenocyte cytotoxicity assays were performed using a modi-
fication of a method previously described (Kimura and Sumiyoshi,
2004). YAC-1 (Target-T) cell line was incubated with BCECF-AM
(molecular probe) at 37 ◦C for 30 min with gentle agitation. BCECF-
AM labelled YAC-1 cells were washed twice with RPMI-1640
medium for cytotoxicity assays. The splenocytes and BCECF-AM
labelled YAC-1 cells were placed in V-bottom 96-well plates at the
ratio of effector (E):target (T) of 25:1; 50:1, and 100:1 and incu-
bated for 4 h; next, these cell mixtures were centrifuged at 410 × g
for 10 min. The total fluorescence intensity of BCECF labelled YAC-
1 cells (Target-T) was determined after adding 1.0% Triton X-100
for lysing. The fluorescence intensity of the supernatant was mea-
sured using a TRAID LT ELISA reader (Dynex technology, VA, USA)
with excitation at 485 nm and emission at 535 nm. The splenocytes
cytotoxicity activity was calculated as follows: percent specific
cytotoxicity = (fluorescence intensity of target cell treated with
splenocytes that was isolated from the experimental group flu-
orescence intensity of spontaneous release of target cells)/(total
fluorescence intensity of target fluorescence intensity of sponta-
neous release of target cells) × 100.
Immunohistochemical analysis
The formalin-fixed CT26 tumours were paraffin-embedded, and
tumour sections were analyzed using haematoxylin–eosin stain-
ing, and were immune-stained with anti-mouse CD8 (Abbiotech,
CA), anti-Asialo GM1 (Wako, Osaka, Japan), or anti-mouse PCNA
(SantaCluz, CA) antibody for cytotoxic T cell, NK, or PCNA stain,
respectively. The immunohistochemical stain using the methods
described by Goldstein and Watkins (2008).
Statistical analysis
The results are expressed in this paper as means ± standard
deviation (S.D.). All experimental data were analyzed using one-
way analysis of variance with the Dunnett’s test. Values of p < 0.05
were considered statistically significant.
Results
Characteristic gene expression profiles of AG-treated murine RAW
264.7 macrophage cells
DNA microarray analysis was performed to investigate the
effects of AG on cell physiology using murine RAW 264.7 cell line as
the model. After 6 h of AG stimulation, 534 genes exhibited signif-
icant expression, and 478 genes demonstrated significantly lower
levels of expression (Fig. 2A). A summary of the cytokine genes
(Fig. 2B), chemokine genes (Fig. 2C), and co-stimulatory recep-
tor genes (Fig. 2D) that were differentially up-regulated in the AG
treated cells compared with untreated cells is provided in Fig. 2A–D.
The AG stimulation demonstrated strong immuno-modulatory
properties to the RAW 264.7 cells. The gene profiling analysis
revealed the modulation of several genes including cytokines (e.g.,
IL-1␣, IL-1␤, IL-6, TNF-␣, IFN-␤1 and G-CSF), chemokines (e.g.,

4. 650 L.-C. Yang et al. / Phytomedicine 21 (2014) 647–655
Fig. 2. Microarray analysis of AG on murine RAW264.7 cells. (A) The volcano plot of normalized data of AG treated samples compared to untreated samples. Standard
selection criteria to identify differentially expressed genes are established at |fold change| 1 and P-value <0.05. List of significant changed (B) cytokine, (C) chemokines and
(D) co-stimulatory receptor genes in RAW264.7 cells with AG treatment compared to control. The changes of listed genes were more than 2-fold to control.
CCL3, CCL4, CCRL2, CXCL2 and CXCL10), as well as several co-
stimulatory molecules (e.g., CD14, CD40, CD69, CD82, CD83 and
CD274).
Effects of AG on body weight and spleen weight of mice in innate
immunity experiment
Table 1 indicates the initial and final body weight of the mice in
the innate immunity experiment. No variance in body weight was
initially evident between the groups of mice. The body weight of
the AG group (15 mg/kg) decreased slightly compared to the con-
trol after 3 weeks of administration. However, the body weight
of both AG-treated groups demonstrated no significant difference
from the control group. The spleen plays a major role in the immune
system (Blalock, 1984), and compared to the control group, the
spleen weight of the AG-treated groups increased markedly after
AG administration (5 and 15 mg/kg) (Table 1).
Measurements of splenocytes proliferation from AG-treated mice
in innate immunity experiment
The effect of AG on splenocyte proliferation with and without
mitogens (LPS or ConA) is demonstrated in Fig. 3A. The spleno-
cyte proliferation without mitogen stimulation was significantly
higher in both the AG-treated groups (5 and 15 mg/kg) compared
to control group. In the presence of ConA and LPS, the proliferation
of splenocytes increased significantly compared to the treatments
without mitogen. Compared with normal mice, the proliferation
of splenocytes stimulated with LPS increased significantly in the
mice administrated with AG (5 and 15 mg/kg) in a dose-dependent
manner. However, no evidence of differences between the ConA-
induced splenocyte proliferation was found among the groups.
NK cytotoxicity in AG-treated mice in innate immunity experiment
Fig. 3B illustrates the splenocyte cytotoxicity activities against
the YAC-1 cell of mice after 3 weeks of treatment. No difference in
splenocyte cytotoxicity existed among the groups at an E/T ratio
of 25. At an E/T ratio of 50, the cytotoxicity of the saline-treated
mice, and the AG low-dose (5 mg/kg) treated splenocytes were
similar. However, the AG low dose (5 mg/kg) treated splenocytes
increased the cytotoxicity at a high ratio of effecters (E/T = 100). By
contrast, mice treated with a high dose of AG (15 mg/kg) signifi-
cantly demonstrated enhanced splenocyte cytotoxicity at both E/T
ratios of 50 and 100. Based on this result, AG was found to enhance
splenocytes cytotoxicity against YAC-1 in vivo.
Flow cytometry data of innate immune mice
The AG treatments (5 and 15 mg/kg) significantly stimulated the
percentage of CD3+CD8+ Tc among total splenocytes in a dose-
dependent manner (Fig. 3C). For the ratio of Tc in splenocytes,
the AG-treated groups had 13.9% (5 mg/kg) and 26.6% (15 mg/kg)
greater than control group. However, the percentage of CD3+CD4+
Th cells was not influenced by the AG (16.0 ± 1.6% in saline group;
15.8 ± 1.5% in at 5 mg/kg; 15.1± 1.6% in at 15 mg/kg). Both AG

5. L.-C. Yang et al. / Phytomedicine 21 (2014) 647–655 651
Table 1
Innate immunity effects of AG on body weight, spleen weight in BALB/c mice.
Group Dose (mg/kg) Initial body weight (g) Final body weight (g) Spleen weight (g)
Control – 27.7 ± 1.4 28.5 ± 2.0 0.12 ± 0.02
AG 5 27.8 ± 1.1 28.0 ± 1.1 0.17 ± 0.02***
AG 15 27.2 ± 1.0 27.6 ± 1.1 0.18 ± 0.03***
***
Values were significantly different compared with the control group by Dunnett’s test: p < 0.001.
treatments (5 and 15 mg/kg) induced the percentage of mature DC
(CD86+MHCIIhigh) in each sample slightly (Fig. 3D).
Effects of AG on body weight, spleen weight and tumour growth of
CT26-bearing mice
The initial body weight of the mice was 22–24 g and no variance
existed among groups. After receiving treatment for 3 weeks, the
body weight of CT26-bearing mice significantly decreased in the
5-FU treated group compared to the saline-treated group. How-
ever, the AG treatment did not influence the body weight in mice
compared to the saline-treated group (Table 2). The weight of the
spleens of mice bearing CT26 tumours significantly increased by
54% compared to the control group. The spleen weight of CT26-
bearing mice administrated with AG (15 mg/kg) was 1.25 times
higher than the saline-treated group. However, 5-FU treatment
introduced a 57.0% decrease in spleen weight, a significant differ-
ence from the spleen weight of the saline-treated groups (Table 2).
Fig. 4 demonstrates changes of tumour sizes from Day 10 to Day 20.
Both AG (15 mg/kg) and 5-FU treatments significantly inhibited the
growth of implanted CT26 tumours. Administration of AG and 5-FU
for 3 week led to a 61.7% (AG-15 mg/kg) and 69.8% (5-FU) decrease
in the tumour size.
Flow cytometry data of CT26 bearing mice
The immuno-modulation of AG was tested using flow cytometry
analyses of splenic cells isolated from CT26-bearing mice. Fig. 5A
indicates that among all the splenocytes, the DC cells (CD11c+)
increased significantly after AG treatments. No significant differ-
ence in the number of CD11c+ cells was evident between the control
and CT26 bearing group. The result of the CD11c+CD137+ expres-
sion of DC was similar to the percentage of CD11c+ expression in
the splenic cells. The intraperitoneal AG injection treatments of the
CT26 bearing mice not only significantly increased the percent-
age of CD11c+ DC, but also the number of CD11c+CD137+ DC. The
expressions of CD11c+ and CD11c+CD137+ splenocytes in the CT26
bearing mice were not influenced by 5-FU compared to the saline
group.
Fig. 3. Innate immunity effects of AG in BALB/c mice. (A) Proliferation of splenocytes induced by mitogen, Con A or LPS; (B) splenic NK cytotoxicity against YAC-1 cells in
AG-treated mice; (C) percentage of CD3+
CD8+
cells among total splenocytes from AG-treated or control mice; (D) percentage of CD11c+
CD86+
MHCIIhigh
DC among total
splenocytes from AG-treated or control mice. All values were mean ± SD (n = 8). Values were significantly different compared with the control group by Dunnett’s test:
*p < 0.05, **p < 0.01 and ***p < 0.001.

6. 652 L.-C. Yang et al. / Phytomedicine 21 (2014) 647–655
Table 2
Anti-tumour effects of AG on body weight, spleen weight and tumour weight in CT26-bearing mice.
Treatments Dose (mg/kg) Final body weight (g) Spleen weight (g) Tumour weight (g)
Control
–
– 24.5 ± 1.0 0.10 ± 0.01 –
CT26 bearing + Saline 26.5 ± 1.2 0.16 ± 0.05#
1.18 ± 0.23
CT26 bearing + AG 5 25.3 ± 1.1 0.17 ± 1.1 0.63 ± 0.29***
15 26.6 ± 1.5 0.21 ± 0.04*
0.49 ± 0.16***
CT26 bearing + 5-FU 25 20.7 ± 1.9***
0.09 ± 0.01**
0.44 ± 0.25***
#
Values were significantly different compared with the control group by Dunnett’s test: p < 0.05.
*
Values were significantly different compared with the CT26 bearing-saline group by Dunnett’s test: p < 0.05.
**
Values were significantly different compared with the CT26 bearing-saline group by Dunnett’s test: p < 0.01.
***
Values were significantly different compared with the CT26 bearing-saline group by Dunnett’s test: p < 0.001.
The percentages of CD3+CD8+ splenic cells are indicated in
Fig. 5B. The presence of CT26 tumour did not influence the per-
centage of CD3+CD8+ Tc compared to control group. AG treatment
administered to CT26 bearing mice significantly increased the per-
centage of CD3+CD8+ Tc of total splenocytes compared to the saline
group. As for CD3+CD4+ Th analysis, CT26 bearing would not affect
the percentage of Th compared to the control group. Although
the percentage of CD3+CD4+ Th cells increased with AG treat-
ment, no significant difference compared to the saline group was
observed (17.1 ± 0.5% in CT26 saline group; 17.7 ± 0.6% in CT26-AG
at 5 mg/kg; 17.8 ± 0.5% in CT26-AG at 15 mg/kg). A 5-FU treatment
administered to CT26-bearing mice slightly decreased the number
of CD3+CD8+ cells, and demonstrated no significant difference from
the saline group.
Fig. 5C illustrates the percentage of CD49b+CD3e− cells of
the total splenocytes in the control and CT26-bearing mice
groups. The bearing of CT26 tumours did not change the per-
centage of CD49b+CD3e− cells compared to the control group.
AG treatments stimulated the number of NK cells in CT26-
bearing mice in a dose-dependent manner, but only a high dose
of AG (15 m/kg) demonstrated significant increases compared
to the CT26-saline group (19.4% increase in AG 15 mg/kg than
saline group). However, 5-FU down-regulated the number of
CD49b+CD3e− splenocytes.
Fig. 4. Intraperitoneal injection of AG and 5-FU inhibit the growth of implanted
CT26 tumours in BALB/c mice. Tumour growth curve was shown and tumour volume
was scored every 3 days from day 7 to day 20. Values were significantly different
compared with the CT26-saline group by Dunnett’s test: **p < 0.01 and ***p < 0.001.
Splenocytes cytotoxicity in CT26-bearing mice
The splenocyte cytotoxic activities against the YAC-1 cell in
the CT26-bearing mice and the control group are indicated in
Fig. 5D. CT26 bearing-saline demonstrated no difference in spleno-
cyte cytotoxicity from the control group, but AG treatments in
CT26-bearing mice stimulated the cytotoxicity activity in a dose-
dependent manner among E/T ratios at 25, 50, and 100. The
splenocyte cytotoxicities in the 5-FU treated CT26-bearing mice
were lower than in the saline-treated mice without significant vari-
ance among any of the E/T ratios.
Immunohistochemistry stain on tumour
The number of infiltrate NK- and Tc-positive cells in the implan-
tation tumour was increased by AG treatment in a dose-dependent
manner, as demonstrated in Fig. 6. These findings indicate that the
immune function of the tumour may be stimulated by the admin-
istration of AG (5 and 15 mg/kg, i.p.). Furthermore, the number of
PCNA-positive cells was widespread in the CT26 tumour slices, and
AG significantly reduced the number of PCNA-positive cells in the
tumour (Fig. 6).
Discussion
In this study, we demonstrated the immune-stimulating activity
of AG in vivo, as well as in vitro. In addition, AG treat-
ments demonstrated potential immuno-modulatory in innate and
tumour-bearing mice. Finally, the anti-tumour effects generated by
AG treatments may be through both Tc and NK activities.
DNA microarray analysis was performed to investigate the dif-
ferential effects of AG treatments. AG stimulation provided strong
immuno-modulatory properties to RAW 264.7 cells. Among the
up-regulated gene expression, IL-1␣, TNF-␣, CD69 and CD83 were
related to anti-tumour activities. IL-1␣ is a member of the IL-1
cytokine family, and is responsible for the pathogenesis of chronic
inflammatory disease (O’Neill, 1995). Active macrophage, neu-
trophils, epithelial cells and endothelial cells are the major cells
to produce IL-1␣. TNF-␣ is a multifunctional cytokine and acts
in anti-tumour activity, immune modulation, inflammation and
haematopoiesis (van Horssen et al., 2006). In addition, TNF-␣
is used in clinical trials as an anti-tumour agent (van Horssen
et al., 2006). AG up-regulated the expression of CD69, known as
an activator molecule, and a differentiation antigen on T and B
cells, macrophages, neutrophils, eosinophils and NK cells (Borrego
et al., 1999). Borrego et al. (1999) demonstrated that CD69 is a
stimulatory receptor for natural killer cells, and that it mediated
NK cytotoxicity. The microarray analysis of AG indicated that AG
was a potent immuno-modulator and may affect the functions
of macrophage, T lymphocytes, and NK. CD83 is expressed at the

7. L.-C. Yang et al. / Phytomedicine 21 (2014) 647–655 653
Fig. 5. Effects of AG and 5-FU in CT26-bearing mice (A) percentage of CD3−
CD11c+
DC (grey bar, left y-axis) and CD3−
CD11+
CD137+
DC (black bar, right y-axis) among total
splenocytes from CT26-bearing mice; (B) percentage of CD3+
CD8+
cells among total splenocytes from CT26-bearing mice; (C) percentage of CD49b+
CD3−
NK among total
splenocytes from CT26 bearing; (D) splenic NK cytotoxicity against YAC-1 cells in CT26-bearing mice. All values were mean ± SD (n = 8). Values were significantly different
compared with the CT26-saline group by Dunnett’s test: *p < 0.05, **p < 0.01 and ***p < 0.001.
surface of most DC and up-regulated together with co-stimulatory
molecules, such as CD80, and CD86 during the maturation of DC
(Fujimoto and Tedder, 2006). CD83 is not only a marker for DC
maturation but is also an essential molecule for CD4+ T cell gen-
eration, and is one of the most important regulatory molecules in
the immune system (Fujimoto and Tedder, 2006). CD83 has been
reported to have the ability to potentiate anti-tumour immunity in
mice-implanted melanoma (Scholler et al., 2002).
Polysaccharides isolated from plants or microorganisms are
used as complementary and alternative medicine in Asian
countries. Recently, numerous studies have demonstrated that
polysaccharides are potent immuno-modulators that can stimu-
late innate immune in vivo and in vitro (Tzianabos, 2000). In this
study, we demonstrated that intraperitoneal injections of AG could
stimulate potent innate immunity in BALB/c mice. The AG treat-
ments significantly increased the spleen weight of mice. However,
the body weight of the mice was not affected by AG. After 3 weeks
of AG treatment the level of AST, APT and total bilirubin was not
influenced (data not shown), indicating that AG did not cause any
obvious toxicity in mice. The spleen plays a central role in regu-
lating the immune system (Tarantino et al., 2011). The immune
function of the spleen modulates the immune system through
phagocytosis, cytotoxicity, and also through T cell-mediated immu-
nity and B cell-mediated humoral immunity (Tarantino et al., 2011).
Our results indicated that AG treatments improved the effect of
splenocytes proliferation, which was successively promoted in a
dose–response manner. The splenocyte proliferation activity was
induced by AG co-treated with LPS, rather than with ConA. Both
cellular and humoral immunity were included in this immune
response, influenced T cells and B cells respectively, and played a
substantial role in the host defence system. The host defence immu-
nity is linked with body responses, such as tumours, inflammation,
and fever (Liu et al., 2012). The ability to develop an effective T and
B cell immunity can be regarded as a stimulation of the lympho-
cyte proliferation response (Liu et al., 2012). Our results indicated
that AG co-treated with LPS stimulated splenocyte proliferation
and demonstrated that AG could significantly increase the activate
potential of B cells, and enhance the humoral immunity.
In this study, we demonstrated that AG enhanced the cyto-
toxicity of splenic NK cells against YAC-1 cells in both innate
immune mice and tumour bearing mice. NK cells play a cen-
tral role in the immune defence system. The immunotherapy of
cancer was dependent on the activation of NK cells (Kim et al.,
2007). Therefore, the results suggested that splenic NK cells were
activated by AG treatment and had beneficial anticancer effects.
Numerous polysaccharides isolated from plants and mushrooms
have been demonstrated to enhance NK cytotoxicity. Hauer and
Anderer (1993) demonstrated that the arabinogalactan from larch
could enhance NK cytotoxicity, not by initiating it directly, but
through the cytokine network. In addition, a mushroom derived
from ␤-glucan Krestin has been reported to inhibit tumour growth
through its stimulation of CD8+ T cells and NK cells (Lu et al., 2011).
A polysaccharide derived from the mushroom Grifola frondosa
appears to be a NK cell stimulator that responds to the anti-tumour
immunity (Kodama et al., 2005). We also measured the popula-
tion of CD49b+CD3− NK cells among splenocytes in tumour-bearing

8. 654 L.-C. Yang et al. / Phytomedicine 21 (2014) 647–655
Fig. 6. Effects of intraperitoneally (i.p.) administered AG on (A) natural killer cell-, (B) cytotoxic T cell-, or (C) PCNA-positive cell numbers in implanted CT26 tumour of mice.
The arrows indicate the positive stained cells.
mice, and found that NK cells significantly increase in number with
AG treatment. An IHC study also replicated the results and managed
to AG increase the presence of NK cells in tumour. AG treatment
made the NK cell filtration in tumours visible. Their results sug-
gested that the anti-tumour effects of AG included increasing the
number of NK cells and promoting cytotoxicity.
Lucas et al. (2007) reported that NK cell priming needs the
presence of CD11chigh DC. DC can also trigger antibody and NK
cell responses, which may contribute to tumour immunity. In
this study, we found that the population of CD11c+ DC among
splenocytes was up-regulated by AG in an innate immunity experi-
ment with tumour-bearing mice. DCs are major antigen-presenting
cells that stimulate innate and adaptive immunity (Meylan et al.,
2006). Anti-tumour immunity originates from DCs, and is central
to tumour antigen presentation, and T-cell activation (Grivennikov
et al., 2010). The TNF receptor family member CD137 plays a role in
inducing immature DC to mature DC (Kwajah and Schwarz, 2010).
In addition, CD137 ligand promotes the maturation of immature
DC, resulting in an induced expression of MHC Class II, cytokines,
and the ability of DC to migrate (Kwajah and Schwarz, 2010). In
our results, AG increased the percentage of CD11c+CD137+ DC in
tumour-bearing mice. The mature DC loaded tumour-antigen had
to generate protective T-cell responses including the production of
CD8+ Tc cells with cytotoxic potential (Mellman et al., 2011). Yeast-
derived ␤-glucans have been reported to have an anti-tumour
function by activating DC and macrophages (Qi et al., 2011). Masuda
et al. (2013) reported that G. frondosa derived ␤-glucan can induce
DC maturation, and has a therapeutic anti-tumour response. The
expression of CD11c+ CD137+ DC could be included in anti-tumour
responses in this study.
CD8+ cytotoxic T cells also play an important role in anti-tumour
immunity (Mellman et al., 2011). Clinicopathological studies have
demonstrated a strong association between prolonged patient sur-
vival and the presence of intra-tumoural CD3+ or CD8+ Tc cells
(Mellman et al., 2011). In this study, AG treatments not only
increased the number of CD3+CD8+ T cells, but also generated the
polarizing of Tc in both innate and tumour bearing mice. Tc is
cytotoxic to allogenic cells, such as tumour cells or infected cells
(Hariharan et al., 1995). The presence of tumour-filtrating lym-
phocytes has been found to correlate with improved survival in
epithelial ovarian cancer (Zhang et al., 2003). In addition, Sato
et al. (2005) reported that CD8+ tumour-filtrating lymphocytes
were associated with the prognosis of ovarian cancer. The IHC data
indicated that more CD8+ filtrating lymphocytes were observed in
AG-treated mice. The immune modulation of AG, such as NK, DC
and Tc activations, responded to the inhibition of tumour weight
size by AG.
The polysaccharide is regarded as an adjuvant in anti-tumour
immunity. Qi et al. (2011) demonstrated that yeast-derived
␤-glucan activated augmented anti-tumour monoclonal antibody-
mediated therapeutic efficacy, and was effective at prolonging
survival rate and reducing tumour size. The ideal adjuvant or adju-
vant combination is expected to trigger the maturation of DC to a
state where DC can facilitate the generation of tumour-reactive,
CD8+ cytotoxic T cells (Mellman et al., 2011). The anti-tumour
immunity of AG fitted the characters as an ideal anti-tumour adju-
vant.
In this study, we demonstrated that AG purified from A. for-
mosanus is capable of stimulating innate immune and anti-tumour
immunity. The mechanisms of AG reactions on DC, NK and Tc are
currently being studied, but it is clear that AG has the potential to
be developed into an immunotherapeutic drug, or to be used in
adjuvant therapy in future.
Conflict of interest
The authors state no conflict of interest.

9. L.-C. Yang et al. / Phytomedicine 21 (2014) 647–655 655
Acknowledgement
This study was supported by grants from the National Science
Council of the Republic of China (NSC102-2320-B-039-031-MY2)
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