Adjuvant effect of Atractylodis macrocephalae Koidz. Polysaccharides on the immune response to foot-and-mouth disease vaccine

Article abstract of DOI:10.1016/j.carbpol.2011.09.080

Present study was designed to investigate the polysaccharide (RAMPS) extracted from the rhizome of Atractylodis macrocephalae Koidz. (RAM) for its effect on the immune responses to foot-and-mouth (FMD) vaccine. Thirty-five ICR mice were randomly distributed into 5 groups with 7 mice in each. After oral administration of RAMPS for 4 days at a dose of 0, 0.025, 0.05, 0.1 g or 0.25 g of RAM, respectively, the animals were immunized twice with FMD vaccine at 2-week intervals. Three weeks later, serum IgG, IFN-γ/IL-5, splenocyte proliferation, and mRNA expression of cytokines by splenocytes were measured. Results indicated that RAMPS and RAM significantly enhanced IgG titers, IgG subclasses, IFN-γ, IL-5 and the splenocyte proliferations stimulated by concanavalin A, lipopolysacharide, and FMDV (P < 0.05). Therefore, RAMPS and RAM increased both cellular and humoral immune responses to vaccination, and may have a potential as an oral adjuvant to improve vaccination.

Full text of DOI:10.1016/j.carbpol.2011.09.080

Carbohydrate Polymers 87 (2012) 1713–1719
Contents lists available at SciVerse ScienceDirect
Carbohydrate Polymers
journal homepage: www.elsevier.com/locate/carbpol
Adjuvant effect of Atractylodis macrocephalae Koidz. polysaccharides on the
immune response to foot-and-mouth disease vaccine
Feng Xie, Yutao Li, Fei Su, Songhua Hu∗
Department of Veterinary Medicine, College of Animal Sciences, Zhejiang University, Hangzhou, Zhejiang 310058, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Present study was designed to investigate the polysaccharide (RAMPS) extracted from the rhizome of
Atractylodis macrocephalae Koidz. (RAM) for its effect on the immune responses to foot-and-mouth (FMD)
vaccine. Thirty-five ICR mice were randomly distributed into 5 groups with 7 mice in each. After oral
administration of RAMPS for 4 days at a dose of 0, 0.025, 0.05, 0.1 g or 0.25 g of RAM, respectively,
the animals were immunized twice with FMD vaccine at 2-week intervals. Three weeks later, serum
IgG, IFN-␥/IL-5, splenocyte proliferation, and mRNA expression of cytokines by splenocytes were mea-
sured. Results indicated that RAMPS and RAM significantly enhanced IgG titers, IgG subclasses, IFN-␥, IL-5
and the splenocyte proliferations stimulated by concanavalin A, lipopolysacharide, and FMDV (P < 0.05).
Therefore, RAMPS and RAM increased both cellular and humoral immune responses to vaccination, and
may have a potential as an oral adjuvant to improve vaccination.
Received 20 March 2011
Received in revised form
24 September 2011
Accepted 27 September 2011
Available online 4 October 2011
Keywords:
Atractylodis macrocephalae Koidz.
Polysaccharide
Oral adjuvant
© 2011 Elsevier Ltd. All rights reserved.
Foot-and-mouth disease
1. Introduction
of doses, use of different administration route (e.g., intradermal
versus intramuscular administration), accelerating dosing schedule
Foot-and-mouth disease (FMD) is a highly contagious disease
affecting cloven-hoofed animals such as cattle, swine and sheep. It
is one of the most important animal diseases and may exert destruc-
tive socio-economic impacts on the countries affected (Mayr,
O’Donnell, Chinsangaram, Mason, & Grubman, 2001). For example,
the 2010 FMD outbreak in Japan and South Korea caused billions
of dollars of losses (Rome, 2010). Vaccination with inactivated
FMD virus (FMDV) is a major means for the control of this dis-
ease in many countries. However, this inactivated FMD vaccines
do not induce broadly reactive long-term protection (Rodriguez &
Grubman, 2009), and failure to elicit effective immune responses
by vaccination in some areas in China has been reported. For exam-
ples, Tian and He (2008) analyzed 129 serum samples of the pigs
having received vaccination against FMD and found that only 54.2%
of the samples had antibody titers required for immune protection
immunity. Liu, Yang, and Wang (2010) observed only 51.33% of
the pigs had produced immune responses with antibody titer high
enough for protection following vaccination against FMD in Liping
county of Guizhou Province of China. Therefore, searching for new
approaches to the improvement of FMD vaccination is needed.
Strategies to improve the immune response to vaccination have
included the use of higher vaccine dose or increasing number
and use of adjuvants such as antigen delivery systems and vari-
ous immunomodulators (Bryan, Sjogren, Perine, & Legters, 1992;
Disis et al., 1996; Mitwalli, 1996; Rey et al., 2000). There is grow-
ing evidence that medicinal herbs and the ingredients enhance the
immune response to vaccination against infectious agents (Rajput,
Hu, Xiao, & Arijo, 2007; Song, Bao, Wu, & Hu, 2009; Sun, Ye, Pan,
& Pan, 2004; Yang, Sun, & Fang, 2005). Atractylodis macrocephalae
Koidz. is a plant in family of Compositae with a plenty of natural
resource in Zhejiang Provinces in China. The rhizome of the plant
(RAM) has been utilized for at least 2000 years as a traditional Chi-
nese medicine (Chen, He, Jiang, & Qiu, 2007). RAM contains different
active ingredients, such as polysaccharides, volatile oil and lactones
(Duan, Xu, Liu, & Li, 2008). Our previous study has demonstrated
that oral administration of a decoction made from RAM has sig-
nificantly increased immune responses to vaccines against FMD
in mice (Li, Sakwiwatkul, Yutao, & Hu, 2009). But it is not clear
that which ingredient(s) contribute the adjuvant activity. In this
study, polysaccharide (RAMPS) extracted from RAM was investi-
gated for its effect on the cellular and humoral immune responses
to a commercial FMD vaccine in mice.
2. Materials and methods
2.1. Animals
∗
Corresponding author at: 866 Yu Hang Tang Rd, Hangzhou, Zhejiang 310058, PR
China. Tel.: +86 571 88982852; fax: +86 571 88982275.
Female ICR mice (5 weeks old) weighing 18–22 g were pur-
chased from Shanghai Laboratory Animal Center (SLAC) Co. Ltd.
E-mail address: songhua@zju.edu.cn (S. Hu).
0144-8617/$ – see front matter © 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carbpol.2011.09.080

1714
F. Xie et al. / Carbohydrate Polymers 87 (2012) 1713–1719
(Shanghai, China), and housed in polypropylene cages with saw-
dust bedding in hygienically controlled environment with
were harvested for determination of lymphocyte proliferation and
cytokines mRNA expression.
a
temperature of 24 1 ◦C, humidity of 50 10%, and a 12/12 h
light/dark cycle. Feed and water were supplied ad libitum. All pro-
cedures related to the animals and their care conformed to the
internationally accepted principles as found in the Guidelines for
Keeping Experimental Animals issued by the Government of China.
2.5. FMDV-specific IgG and the IgG subclasses
Serum samples were analyzed for measurement of serum IgG
and the isotypes by an indirect double antibody sandwich enzyme-
linked immunosorbent assay. The wells of polyvinyl 96-well
microtiter plates were coated with 50 ␮l rabbit anti-FMDV serotype
O antibody (LVRI, China) diluted in 0.05 M carbonate/bicarbonate
buffer (1:800), pH 9.6, and incubated overnight at 4 ◦C. After five
washes with phosphate buffer saline containing 0.05% Tween-20
(PBST), the wells were blocked with 5% skimmed milk and incu-
bated at 37 ◦C for 2 h. Thereafter, 50 ␮l FMDV type O antigen (LVRI)
(1:8 dilution) was added and incubated at 4 ◦C for 2 h. After wash-
ing, 50 ␮l of serum (diluted serially for IgG or diluted 1:50 for
isotype analysis in PBS 5% skimmed milk) was added to each
well and incubated at 37 ◦C for 1 h. Plates were then washed five
times in PBST. For IgG titer detection, 50 ␮l of goat anti-mouse
IgG horseradish peroxidase conjugate (1:5000) was added to the
wells and incubated at 37 ◦C for 1 h. Plates were washed again with
PBST. Fifty microliters of 3,3ꢀ,5,5ꢀ-tetramethyl benzidine solution
(100 ␮g/ml of 0.1 M citrate–phosphate, pH 5.0) was added to each
well and incubated for 15 min at 37 ◦C. The reaction was stopped
by adding 50 ␮l of 2 M H2SO4 to each well. The optical density of
the plate was read by an automatic ELISA plate reader at 450 nm.
Values above the cut-off background level (mean value of sera
from unimmunized mice multiplied by a factor of 2.1) were con-
sidered positive. Titers were depicted as reciprocal end-dilutions.
For subclasses, 50 ␮l of horseradish peroxidase conjugated goat
anti-mouse IgG1 or IgG2a or IgG2b or IgG3 (1:2000) was added
to corresponding plate and then incubated for 1 h at 37 ◦C.
2.2. Chemicals and reagents
Concanavalin
A
(ConA), lipopolysaccharide (LPS from
Escherichia coli 055:B5) and 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide (MTT) were purchased from Sigma
Chemical Co., USA; goat anti-mouse IgG peroxidase conjugate was
from Kirkegaard & Perry Laboratories, Inc., USA; goat anti-mouse
peroxidase conjugate IgG1, IgG2a, IgG2b and IgG3 were from
Southern Biotech. Assoc., Birmingham, AL, USA; RNAisoTM plus
was from TaKaRa Biotechnology (Dalian) Co., Ltd., China; revert
AidTM M-MuLV reverse transcriptase was from Fermentas, USA;
diethylpyrocarbonate (DEPC) and ribonuclease inhibitor was from
Biobasic, Canada; oligo (dT)18 was from Sangon, China.
2.3. Preparation of RAM decoction and RAM polysaccharides
(RAMPS)
Dried rhizome of Atractylodis macrocephalae Koidz. (RAM) was
purchased from Hu Qing Yu Tang Co. Ltd., Hangzhou, China. The
drug source was in Pan’an County of Zhejiang Province of China
where the best RAM is produced. RAM decoction was prepared
according to method as previously described by Li, Sakwiwatkul,
et al. (2009). Polysaccharide was extracted from RAM as follows.
Briefly, the rhizome (100 g) was ground into powder and then
extracted with boiling water two times under reflux for 2 h each
time. The aqueous portion was filtered through filter paper. The
filtrate was concentrated under reduced pressure, and then cen-
trifuged at 3000 rpm for 15 min. Four volumes of 95% ethanol were
added to the supernatant, and kept overnight at 4 ◦C. The resulting
precipitate was dissolved in distilled water, subjected to Macrop-
orous Adsorption Resin column D101, and then washed with water.
The collected elute was concentrated, dialyzed against distilled
water (cut-off Mw 7000 Da) and lyophilized to afford a total RAM
polysaccharide (RAMPS, light off-white powder, 4.2 g).
Total sugar content was estimated by the phenol–sulfuric
acid analysis using glucose as a standard (Bitter & Muir, 1962).
Neutral monosaccharide composition was analyzed according
to the following procedure: RAMPS (10 mg) were hydrolyzed
with 5 ml of 2 M TFA at 110 ◦C for 6 h to release component
monosaccharides. The hydrolyzed monosaccharides (inositol as the
internal standard) were derivatized to acetylated aldononitriles
(Mawhinney, Feather, Barbero, & Martinez, 1980) and isother-
mally separated by gas chromatography (GC) in an Agilent 6890N
system (Agilent Technologies, Palo Alto, CA, USA) equipped with
a flame-ionization detector (FID) and a DB-5 capillary column
(30.0 m × 0.32 nm × 0.25 ␮m).
2.6. Splenocyte proliferation assay
Spleen was collected 3 weeks after the booster immunization
from the immunized mice under aseptic conditions, and kept in
Hank’s balanced salt solution (HBSS, Sigma). The organ was minced
and passed through a fine steel mesh to obtain a homogeneous
cell suspension. To lyse contaminated erythrocytes, 0.83% NH4Cl
in 0.01 M Tris–HCl (pH 7.2) was added to the suspension. After
centrifugation (380 × g at 4 ◦C for 10 min), the pelleted cells were
washed three times in HBSS and re-suspended in complete medium
(RPMI 1640 supplemented with 0.05 mM 2-mercaptoethanol,
100 IU/ml penicillin, 100 ␮g/ml streptomycin and 10% heat inac-
tivated FBS). Cell numbers were counted with a haemocytometer
by trypan blue dye exclusion method. Cell viability exceeded
95%. Splenocyte proliferation was assayed as described previously
with some modification (Sun et al., 2004). Briefly, splenocytes
were seeded into a 96-well flat-bottom microtiter plate (Nunc)
at 5.0 × 106 cells/ml in 100 ␮l complete medium, thereafter Con A
(final concentration 5 ␮g/ml), LPS (final concentration 7.5 ␮g/ml),
FMDV antigen (final concentration 200 ␮g/ml) or medium were
added giving a final volume of 200 ␮l. The plates were incubated at
37 ◦C in a humid atmosphere with 5% CO2 for 2 or 5 days. All the
tests were carried out in triplicate. The cell proliferation was eval-
uated using MTT method. Briefly, 50 ␮l of MTT solution (2 mg/ml)
were added to each well 4 h before the end of incubation. The plates
were centrifuged (1400 × g, 5 min) and the untransformed MTT was
removed carefully by pipetting. To each well 150 ␮l of a DMSO
working solution (192 ␮l DMSO with 8 ␮l 1 M HCl) was added, and
the absorbance was evaluated in an ELISA reader at 570 nm with a
630 nm reference after 15 min. The stimulation index (SI) was cal-
culated based on the following formula: SI = the absorbance value
for mitogen cultures/the absorbance value for non-stimulated cul-
tures.
2.4. Immunization
Thirty-five female ICR mice were randomly divided into five
groups with 7 mice in each. The animals were subcutaneously
injected twice with 200 ␮l of FMDV type O vaccine (Lanzhou Vet-
erinary Research Institute, China) with 2-week intervals. One day
before each immunization, the mice had already been orally admin-
istered for 4 days with 0.25 ml of 0.89% saline solution, or RAMPS
(0.025, 0.05 or 0.1 g) or RAM decoction (0.25 g). Blood samples were
collected 3 weeks after the booster immunization for detection of
IgG titers, the IgG subclasses and western blot analysis. Splenocytes

F. Xie et al. / Carbohydrate Polymers 87 (2012) 1713–1719
1715
2.7. Quantification of target genes by real-time PCR
specific primers, 1 ␮l (5 ␮M) of target gene and ␤-actin specific
probes. Reaction conditions were the standard conditions for the
TagMan PCR (15 s denaturation at 95 ◦C, 30 s annealing at 60 ◦C)
with 40 PCR cycles. Relative quantification between samples was
achieved by the 2−ꢁꢁCT method (Livak & Schmittgen, 2001) and
calculated by software REST 2005 (gifted by Eppendorf Company),
and is reported as the n-fold difference relative to target gene mRNA
expression in the calibrator group (the group of mice orally admin-
istered with saline).
Splenocytes from the FMDV-immunized ICR mice prepared
as described previously were seeded into a 24-well flat-bottom
microtiter plate (Nunc) at 5 × 106 in 2 ml complete medium, there-
after 100 ␮l FMDV antigen (2 ␮g/␮l) was added. The plates were
incubated at 37 ◦C in a humid atmosphere with 5% CO2. After 15 h
treatment, cells were harvested by centrifugation (380 × g at 4 ◦C
for 10 min), and washed with ice-cold PBS, then subjected to RNA
extraction. Splenocytes (1 × 107) were lysed in 1 ml of RNAisoTM
Plus reagent and the total RNA was isolated according to the man-
ufacture’s protocol. The concentration of total RNA was quantified
by determining the optical density at 260 nm. The total RNA was
used and reverse transcription was performed by mixing 2 ␮g of
RNA with 0.5 ␮g oligo (dT)18 primer in a sterile tube. Nuclease-free
water was added giving a final volume of 12.5 ␮l. This mix was incu-
bated at 70 ◦C for 5 min and chilled on ice for 2 min. Then a solution
containing 4 ␮l of M-MuLV 5× reaction buffer, 2 ␮l of 10 mM dNTP,
20 U of ribonuclease inhibitor, and DEPC-treated water was added
giving a final volume of 19 ␮l and the tubes were incubated for
5 min at 37 ◦C. The tubes then received 200 U of M-MuLV reverse
transcriptase and were incubated for 60 min at 42 ◦C. Finally, the
reaction was stopped by heating at 70 ◦C for 10 min. The samples
were stored at −20 ◦C until further use.
2.8. Determination of serum cytokines by western blot
Serum samples were analyzed for measurement of IFN-␥ and
IL-5 by Western blot analysis. For the determination of IFN-␥, the
serum samples were diluted with the sample buffer (1:50), boiled
for 5 min, and then 10 ␮l of each diluted sample was separated
by 12% sodiumdodecyl sulfate-polyacrylamide gelelectrophoresis
(SDS-PAGE). The proteins were transferred onto the immobilon-
p transfer membrane (Millipore Corporation, USA), the membrane
was washed and then blocked in 5% (skimmed milk in TBS) block-
ing agent for 35 min at 37 ◦C in the incubator shaker. After three
washes in TBST (TBS containing 0.1% Tween-20), the membrane
was incubated with 1:1000 diluted IFN-␥ monoclonal antibody
(sc-5992, Santa Cruz Biotechnology Inc., SantaCruz, CA, USA) for
50 min at room temperature on the rocker platform. The mem-
brane was washed three times in TBST, followed by incubating
with HRP-conjugated goat anti-mouse IgG at a 1:2000 dilution for
50 min at room temperature. After the final wash, the immunoblot
was examined by BeyoECL Plus (Beyotime Biotechnology, China)
according to the manufacturer’s instructions. The membrane was
exposed to an X-ray film, which was later developed. For deter-
mination of IL-5, the serum samples were at a 1:20 dilution, and
incubated with 1:800 diluted IL-5 polyclonal antibody (BS3471,
Bioworld Technology, Inc., USA), followed by the correspond-
ing secondary HRP-conjugated goat anti-rabbit IgG (Proteintech
Group Inc., USA) at a 1:2000 dilution, The other procedures was
the same as described above for the detection of IFN-␥. The
intensity of the resulting bands was quantified by the Quantity
One® 1-D Analysis Software Version 4.6.2 (BioRad, Hercules, CA,
USA).
Relative quantitation of GATA-3, T-bet and cytokines cDNA and
␤-actin message was conducted on ABI 7300 (PE Applied Biosys-
tems, USA). The purpose of the house keeping gene (␤-actin) is
to normalize the PCRs for the RNA added to the reverse tran-
scription reactions and to correct for differences in RT reaction
efficiencies (Bustin, 2000). The PCR was performed in a dual PCR
with primers and TagMan probes for the target gene and ␤-actin
cDNA in the same reaction vessel. The primers and probes for tar-
get gene and ␤-actin (Table 1) were designed using Primer Express
3.0 (PE Applied Biosystems) according to the manufacturer’s direc-
tions and each primer was located in different exons of target genes.
Probes for GATA-3, T-bet and cytokines were detected via a 5ꢀ
labeled with reporter dye (FAM) (Sangon Co., Ltd., Shanghai, China)
and 3ꢀ labeled with quench dye (BHQ-1) (Sangon Co., Ltd., Shanghai,
China), while probes for ␤-actin were 5ꢀ labeled with reporter dye
(HEX) (Sangon Co., Ltd., Shanghai, China). The PCR products were
identified by DNA sequencing.
Amplification was carried out in a total volume of 20 ␮l con-
taining 2 ␮l of 10× PCR buffer, 2 ␮l of MgCl2 (25 mmol/L), 2 ␮l of
dNTPmix (2.5 mM), 0.4 ␮l of Tag DNA polymerase (Takara, China),
2 ␮l of cDNA template, 2 ␮l (5 ␮M) of each target gene and ␤-actin
2.9. Statistical analyses
Data were expressed as means standard deviations (S.D.).
Duncan’s test was used to compare the parameters between groups
by using SPSS 13.0. P values of less than 0.05 were considered sta-
tistically significant.
Table 1
Sequences of primer and probes for quantitative RT-PCR of cytokine and transcrip-
tion factors.
Gene
Primer sequence
3. Results
Forward: 5ꢀ-AGCGGTTCCGATGCCCT-3ꢀ
␤-Actin
Reverse: 5ꢀ-AGAGGTCTTTACGGATGTCAACG-3ꢀ
Probe: 5ꢀ-HEX-TCCTTCTTGGGTATGGAATCCTGTGGC-BHQ-1-3ꢀ
3.1. Isolation, characterization and composition of RAMPS
IL-4
Forward: 5ꢀ-GAGACTCTTTCGGGCTTTTCG-3ꢀ
Reverse: 5ꢀ-CAGGAAGTCTTTCAGTGATGTGG-3ꢀ
Probe: 5ꢀ-FAM-CCTGGATTCATCGATAAGCTGCACC-BHQ-1-3ꢀ
The polysaccharide contained in RAMPS was 86.2% as deter-
mined by phenol–sulfuric acid method. RAMPS showed negative
Fehling’s reagent and iodine–potassium iodide reactions, indi-
cating that it did not contain reducing sugar and starch-type
polysaccharides. UV analysis and triketohydrindene hydrate
reaction indicated that RAMPS was not contaminated with
proteins. GC quantitative analysis with derivatization revealed
that RAMPS was composed of rhamnose (4.92 mol.%), arabinose
(12.25 mol.%), xylose (10.17 mol.%), mannose (24.32 mol.%), glucose
(55.74 mol.%), and galactose (6.65 mol.%) with the molar ratio of
1.00:2.49:2.07:4.94:11.33:1.35.
IFN-ꢀ
GATA-3
T-bet
Forward: 5ꢀ-GCTTTGCAGCTCTTCCTCATG-3ꢀ
Reverse: 5ꢀ-CTTCCACATCTATGCCACTTGAG-3ꢀ
Probe: 5ꢀ-FAM-CTGTTTCTGGCTGTTACTGCCACGGC-BHQ-1-3ꢀ
Forward: 5ꢀ-GGTCAAGGCAACCACGTC-3ꢀ
Reverse: 5ꢀ-CATCCAGCCAGGGCAGAG-3ꢀ
Probe: 5ꢀ-FAM-CGCCCGCCTCTGCTGCACG-BHQ-1-3ꢀ
Forward: 5ꢀ-ATTGCCCGCGGGGTTG-3ꢀ
Reverse: 5ꢀ-GACAGGAATGGGAACATTCGC-3ꢀ
Probe: 5ꢀ-FAM-CTGGGAAGCTGAGAGTCGCGCTCA-BHQ-1-3ꢀ

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F. Xie et al. / Carbohydrate Polymers 87 (2012) 1713–1719
Table 2
Effects of oral administration of RAM and RAMPS on the mean body weight (mean S.D. g) of mice (n = 7).
Group/time
Before immunization*
Before booster*
3 weeks after booster*
Saline
RAM
25.05 1.12
25.12 1.15
29.90 1.66
29.99 2.18
34.26 1.81
34.26 2.53
0.25 g
RAMPS
0.025 g
0.05 g
0.1 g
23.97 1.06
24.71 1.11
24.64 1.33
29.19 1.68
29.04 1.52
30.01 2.30
33.85 2.21
34.05 2.40
34.94 2.44
*
No statistical difference between groups at the same time (P > 0.05).
3.2. Effects of oral administration of RAMPS and RAM on the body
weight of mice
To determine if RAMPS has the negative effect on the growth
performance of mice, the body weight of each animal was mea-
sured before and after immunization. No abnormal behavior and
side effects were found in mice throughout the experiment. There
was no significant difference for the body weight between the mice
administered RAM or RAMPS and the control mice administered
saline solution as indicated in Table 2 (P > 0.05).
3.3. FMDV-specific IgG and IgG isotypes
To determine the effect of oral administration of RAMPS on the
specific antibody responses, mice were orally administered saline
(control) or RAM or RAMPS for 4 days, and then immunized s.c.
twice with 200 ␮l of FMDV vaccine at 2-week intervals. Blood sam-
ples were collected 3 weeks after boosting for measurement of
FMDV-specific IgG titers and the IgG subclasses. Fig. 1 indicates
that FMDV-specific IgG titers were higher in mice orally admin-
istered RAM or RAMPS than in mice administered saline solution
only (P < 0.05). Moreover, mice administered RAMPS at a dose of
0.05 g had almost 10-fold higher FMDV-specific IgG titers (1:2826)
than the control (1:290) (P < 0.05).
Fig. 2 shows that the subclasses IgG1, IgG2a, IgG2b and IgG3 lev-
els were higher in groups administered RAM or RAMPS than in the
control group (P < 0.05). RAMPS at a dose of 0.05 g elicited signifi-
cantly higher IgG1, IgG2a, IgG2b (P < 0.05) but not IgG3 responses
compared to RAM.
Fig. 2. FMDV-specific IgG isotypes. Bars with different letters are statistically dif-
ferent (P < 0.05).
3.5. Effect of RAMPS and RAM on mRNA expression of cytokines
and transcription factors by splenocytes
Oral administration of RAM and RAMPS significantly increased
mRNA expressions of cytokines IFN-␥ and IL-4, and transcription
factors T-bet and GATA-3 (P < 0.05) by splenocytes when com-
pared with the control (Figs. 4 and 5). The rates of increase for
Th1 response IFN-␥ and T-bet were 2.34 (4.45/1.76) and 2.13
(3.57/1.68), respectively, which were numerically higher than
those of Th2 response where 1.74 (2.53/1.46) for IL-4 and 1.58
(2.22/1.4) for GATA-3 were detected.
3.4. Effect of RAMPS and RAM on splenocyte proliferation
3.6. Effect of RAMPS on serum IFN-ꢀ and IL-5 levels
Effects of RAMPS on mitogen- and FMDV-stimulated splenocyte
proliferation in the immunized mice are shown in Fig. 3. When
compared with the control, RAM or RAMPS significantly enhanced
splenocyte proliferative responses to ConA, LPS, or FMDV (P < 0.05).
Among groups, mice administered RAMPS at a dose of 0.05 g were
found to have numerically highest splenocyte proliferation.
Western blot analysis showed that RAMPS at the dose of 0.05 g
significantly increased both IFN-␥ and IL-5 (P < 0.05) in serum of
mice immunized with FMD vaccine (Fig. 6).
Fig. 1. FMDV-specific IgG titers. Bars with different letters are statistically different
(P < 0.05).
Fig. 3. Splenocyte proliferative responses to Con A, LPS and FMDV. Bars with differ-
ent letters are statistically different (P < 0.05).

F. Xie et al. / Carbohydrate Polymers 87 (2012) 1713–1719
1717
4. Discussion
In this study, oral adjuvant effect of RAMPS against FMD vac-
cine in mice has been proved. After oral administration for 4 days
of RAMPS, immunization of a commercial FMD vaccine induced sig-
nificantly higher serum specific IgG and the IgG isotype responses
in association with up-regulated serum IFN-␥ and IL-5. In addition,
RAMPS significantly increased splenocyte proliferative responses
to ConA, LPS and FMDV, as well as mRNA expression of Th1/Th2
cytokines (IFN-␥/IL-4) and transcription factors (T-bet/GATA-3) by
splenocytes.
The rhizome of Atractylodis macrocephalae Koidz. has long been
used as a digestive stimulator in China. Chemically, it contains
three categories including volatile oil, lactones and polysaccharides
(Duan et al., 2008). Volatile oil fraction is rich in sesquiter-
penes and acetylenic compounds, such as atractylon, atractylol,
selina-4(14),7(11)-dien-8-one, ␤-Se-linene, aromaden-drene, etc.
(Li, Wen, Cui, Zhang, & Dong, 2007). This fraction is believed to have
antitumor activities. Zhang, Zhang, Shi, and Liu (2006) observed
that volatile oil from RAM significantly suppressed growth of
Ehrlich ascites tumor and extended the life-span of mice after the
animals were peritoneally administered RAM for 7 days at a dose of
50 or 100 mg/kg. Lactone fraction contains atractylenolide I, II, III,
IV with anti-inflammatory properties (Li, He, Dong, & Jin, 2007). Li,
He, and Jin (2007) discovered that atractylenolides I and III inhib-
ited the production of TNF-␣ and NO in peritoneal macrophages
stimulated with LPS in a dose-dependent manner. However, nei-
ther volatile oil nor lactones have been found adjuvant properties
in our experiments (data not shown). Therefore, the adjuvant activ-
ities of RAM may be attributed to the fraction of polysaccharide
(RAMPS).
Fig. 4. Expression of IFN-␥ and IL-4 mRNA by splenocytes. Bars with different letters
are statistically different (P < 0.05).
Many plant-derived polysaccharides have been found to have
immunomodulatory activities, such as the polysaccharides isolated
from Astragalus membranaceus (Li, Chen, Wang, Tian, & Zhang,
2009), Actinidia eriantha (Sun, Wang, Xu, & Ni, 2009), Acanthopanax
senticosus (Shen et al., 1991), and Angelica sinensis (Yang, Jia, Meng,
Wu, & Mei, 2006). RAMPS was composed of rhamnose, arabi-
nose, xylose, mannose, glucose, and galactose with molar ratios
of 1.00:2.49:2.07:4.94:11.33:1.35. RAMPS has a backbone con-
structed by glucose, and the branches constituted by rhamnose
Fig. 5. Expression of T-bet and GATA-3 mRNA by splenocytes. Bars with different
letters are statistically different (P < 0.05).
Fig. 6. Serum IFN-␥ and IL-5 levels measured by western blot. Mice were orally administered saline (control) or RAMPS at a dose of 0.05 g for 4 days. (A) 10 ␮l of each
diluted serum sample (1:50) was used for the determination of IFN-␥, lanes 1–4, control; lanes 5–8, RAMPS. (B) 10 ␮l of each diluted serum sample (1:20) was used for the
determination of IL-5, lanes 1–4, control; lanes 5–8, RAMPS. IFN-␥ (C) and IL-5 (D) were quantified by the Quantity One Software. *P < 0.05 between two groups.

1718
F. Xie et al. / Carbohydrate Polymers 87 (2012) 1713–1719
and mannose (Liang, Guo, & Zhang, 2007). The polysaccharide
isolated from the roots of A. membranaceus (APS) has been inten-
sively studied for its immunomodulatory activities in China (Kang
et al., 2010; Li, Chen, et al., 2009; Zhang et al., 2010). It con-
tains rhamnose, glucose, galactose, arabinose with molar ratios
of 1.19:72.01:5.85:20.95, and the main chain was composed of
glucose (Li, Zhao, & Lv, 2008). Although RAMPS and APS have dif-
ferent sugar composition, many investigations have shown that
polysaccharide structure has influence on biological activity more
than the sugar composition (Deters, Lengsfeld, & Hensel, 2005;
Sakagami et al., 2005; Zhao, Kan, Li, & Chen, 2005). Therefore, simi-
lar molecular structures of RAMPS and APS may contribute to their
immunomodulatory effects.
In addition to promoting an appropriate immune response, an
ideal adjuvant must be less toxic. Medicinal herbs are traditionally
administered via oral route, and oral use of immunomodulators can
avoid the side effects found in parenteral administration (Chavali
& Campbell, 1987; Maharaj, Froh, & Campbell, 1986). As indicated
in Table 2, there was no significant difference of the body weight
between groups, and no abnormal behavior and side effects were
found throughout the experiment. Therefore, RAMPS could be safe
for oral administration.
The efficacy of vaccination against FMD is generally evaluated by
humoral immune response (McCullough et al., 1986, 1992; Yadav,
Sharma, & Chhabra, 2005). As shown in Fig. 1, oral administra-
tion of RAMPS significantly increased serum FMDV-specific IgG
titers in mice, which indicated that RAMPS was able to improve
humoral immunity when used as an adjuvant in FMD vaccine.
Polysaccharide-enhanced humoral immune response has also been
found in other study. Sun and Liu (2008) observed a significantly
increased OVA-specific IgG levels in mice immunized with OVA
in combination with polysaccharide derived from the mycelium
of Polyporus albicans. Cellular immune response, mediated by T
lymphocytes, plays an important role in the host response to intra-
cellular pathogens. An increasing evidence suggested that T cell
mediated immunity is required for protection against FMD in ani-
mals (Collen, Pullen, & Doel, 1989). The capacity to elicit an effective
T cell immunity can be measured by lymphocyte proliferation
response (Marciani et al., 2000). Fig. 3 shows that RAMPS signif-
icantly enhanced ConA-, LPS- and FMDV-stimulated splenocyte
proliferation in the immunized mice, suggesting that both T and
B cells were activated.
Enhanced production of all IgG subclasses may be explained by
increased release of Th1 and Th2 cytokines. Fig. 4 shows that the
mRNA expression of IFN-␥ (Th1 cytokine) and IL-4 (Th2 cytokine) in
splenocytes of the immunized mice were both enhanced by RAMPS,
which may be associated with simultaneously up-regulated mRNA
expression of T-bet and GATA-3 in splenocytes (Fig. 5). Further-
more, a significant increase of IFN-␥ and IL-5 has been observed in
serum from the immunized mice by Western blot analysis (Fig. 6).
All of these results suggested that RAMPS also can modulate the
quality of the immune responses and promote a balanced Th1/Th2
immune responses against FMD in mice.
The oral administration of 0.05 g RAMPS shows the strongest
improvement on the immune response to FMDV but not the largest
dose 0.1 g as indicated in Figs. 1–5. Many herbal extracts show
a dose-dependent effect on the immune responses. For example,
Sun et al. (2009) observed the highest serum OVA-specific anti-
body titers at a dose of 50 ␮g AEPS when the polysaccharides from
the roots of A. eriantha (AEPS) (25, 50, 100 ␮g) administered subcu-
taneously with OVA (100 ␮g) in mice; Song et al. (2009) reported
the highest IgG response in mice immunized with FMDV antigen
plus 10 ␮g of GSLS (ginseng stem-leaf saponin) when mice were
subcutaneously immunized with FMDV antigen with GSLS (1, 5, 10
and 20 ␮g). The exact mechanisms behind this phenomenon are
unclear, but the reasons might be activation of suppressor T cell (Ts
cells) at the high dose or presence of cytotoxic contaminants in the
herbal extracts.
In conclusion, the fraction of polysaccharide in RAM may
contribute to the oral adjuvant activity of RAM. After oral admin-
istration of RAMPS, a FMD vaccine induced significantly higher
specific IgG and the IgG isotype responses in association with up-
regulated serum IFN-␥ and IL-5. Administration of RAMPS also
significantly increased splenocyte proliferative responses to ConA,
LPS and FMDV, as well as mRNA expression of Th1/Th2 cytokines
(IFN-␥/IL-4) and transcription factors (T-bet/GATA-3) by spleno-
cytes. Considering the oral adjuvant activities of RAMPS in FMD
vaccination, its effect on the intestinal mucosal immunity should
be investigated in our further study.
Acknowledgement
The work was supported by the National Natural Science Foun-
dation of China (No. 30972207).
Immunity to different infectious agents requires distinct types
of immune responses. Th1 immune response is required for
protective immunity against intracellular infections, which is
characterized by the production of cytokines IL-2, TNF-␣ and
IFN-␥, and an enhanced production of IgG2a, IgG2b and IgG3
in mice. On the other hand, Th2 immunity is effective for pro-
tection against most bacterial as well as certain viral infection,
which is characterized by the production of cytokines IL-4, IL-
5 and IL-10, and an increased production of IgG1 (Constant &
Bottomly, 1997; Del Prete, De Carli, Ricci, & Romagnani, 1991;
Fiorentino, Bond, & Mosmann, 1989; Livingston et al., 1994). As
shown in Fig. 2, RAMPS not only significant enhanced FMDV-
specific IgG1 level in mice immunized with FMD vaccine, but also
increased specific serum IgG2a, IgG2b and IgG3 levels, indicated
that both Th1 and Th2 immune responses were activated. Simi-
lar results of plant polysaccharides have been reported by several
workers (Lai et al., 2010; Sun & Liu, 2008; Sun et al., 2009). Sun
et al. (2009) observed a significantly increased OVA-specific IgG1,
IgG2a and IgG2b antibody titers in the immunized mice when OVA
was administered subcutaneously with the water-soluble polysac-
charide from the roots of A. eriantha. Lai et al. (2010) reported that
a polysaccharide extract of Ganoderma lucidum induced significant
levels of IgG1 and IgG2a antibodies specific for tetanus toxoid when
immunized intramuscularly with tetanus toxoid in mice.
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