Chinese Medicine, 2011, 2, 178-185
doi:10.4236/cm.2011.24028 Published Online December 2011 (http://www.SciRP.org/journal/cm)
Long-Term Treatment with a Compound
Polysaccharide-Based Health Product (Infinitus Polysac
Plus) Enhances Innate and Adaptive Immunity in Mice
Hoi-Yan Leung1, Chung-Wah Ma2, Qing Tao Tang2, Kam-Ming Ko1*
1Division of Life Science, Hong Kong University of Science & Technology, Hong Kong SAR, China
2Lee Kum Kee Health Products Group Ltd., Hong Kong SAR, China
*
Received September 16, 2011; revised November 10, 2011; accepted November 24, 2011
Abstract
This study aimed to investigate the effects of a compound polysaccharide-based health product (Infinitus Po-
lysac Plus, IPP) on innate and adaptive immunity in mice. Both ex vivo/in vivo mouse models and an in vitro
system using cultured mouse splenocytes were adopted for the assessment of innate and adaptive immunity.
For the innate immune response, long-term IPP treatment (0.26 and 0.78 g/kg  20 doses) enhanced the car-
bon clearance activity and phagocytic rate of macrophages, as well as natural killer cell activity in mice. The
IPP-induced increase in natural killer cell activity was accompanied by the suppression of tumor growth in
Sarcoma-180 cell-inoculated mice. For the adaptive immune response, while long-term IPP treatment in-
creased the splenocyte index in mice, IPP incubation with mouse splenocytes in vitro potentiated their con-
canavalin A-stimulated proliferation. Long-term IPP treatment significantly restored the hemolytic activity
of serum on sheep red blood cells and dinitrofluorobenezene-induced delayed-type hypersensitivity in sensi-
tized and immunosuppressed mice. In conclusion, the results indicate that long-term IPP treatment produces
stimulatory effects on both innate and adaptive immunity in mice.
Keywords: Compound Polysaccharide, Innate Immunity, Adaptive Immunity
1. Introduction
Polysaccharides isolated from medicinal fungi and herbs
are known to possess immunomodulatory activity in both
experimental and clinical settings [1,2]. It has been sug-
gested that a mixture of polysaccharides from various
medicinal fungi and herbs can produce immunomodula-
tory effects in a synergistic manner [3-5]. Innate and adap-
tive immunity are the mainstay of immune defense against
microbial infections. While innate immune responses,
which involve phagocytotic activities and secretion of
pro-inflammatory mediators, do not require prior expo-
sure to an antigen, adaptive immune responses, such as the
eliciting of antigen-specific cell-mediated response and pro-
duction of antigen-specific antibodies, are dependent on
processes generated by previous exposure to an antigen
[6,7]. In the present investigation, we aimed to define the
immunopharmacological profile of Infinitus Polysac Plus
(IPP), a Chinese herbal health product containing a mix-
ture of biologically active polysaccharides, on innate and
adaptive immunity in mice, using both ex vivo/in vivo
mouse models and an in vitro system utilizing cultured
mouse splenocytes. IPP has been consumed by hundreds
of thousands of individuals over the past 15 years in
China.
2. Materials and Methods
2.1. Chemicals, Cell Materials and Herbal
Product
Concanavalin A (Con A), 2,4-dintro-1-fluorobenzene
(DNFB) and cyclophosphamide (CYC) were purchased
from Sigma (St. Louis, MO). RPMI-1640 medium and
fetal calf serum were obtained from GibcoTM (Grand
Island, NY). MTT (3-[4, 5-dimethyl-thiazol-2-yl]-2,5-di-
phenyl tetrazolium bromide)-based cell proliferation kit I
was purchased from Boehrimger Mannheim Gmbh (Ger-
many). Chicken red blood cells (CRBC) and sheep red
blood cells (SRBC) were purchased from Hemostat La-

179 H.-Y. LEUNG ET AL.
boratories (Dixon, CA). YAC-1 cells and Sarcoma 180
cells were supplied by American Type Culture Collec-
tion (ATCC; Manassas, VA). All other chemicals were
of analytical grade.
IPP was prepared by mixing the individual aqueous ex-
tract of 9 herbs, namely, Fructus Lycii [Lycium barba-
rum L. (Solanaceae)], Poria [Poria cocos (Schw.)] Wolf,
Flos Chrysanthemi (Chrysanthemum morifolium Ramat.),
Radix Codonopsis [Codonopsis pilosula (Franchet)
Nannfeldt (Campanulaceae)], Lentinula [Lentinus edodes
(Berk.) Sing. (Agaricaceae)], Fructus Mori (Morus alba
L.), Hericium (Hericium erinaceus), Flammulinae (Fla-
mmulina velutipes) and Tremella (Tremella fuciformis).
All herbs were authenticated with reference to morpho-
logical and chemical criteria stated in Chinese Pharma-
copoeia. The combined extract was dried and made into
a tablet dosage form. The total polysaccharide content of
IPP was 15% (w/w) as determined by a colorimetric me-
thod [8].
2.2. Animal Care
Adult female and male ICR mice were maintained under
a 12-h dark/light cycle at an ambient temperature of ap-
proximately 22˚C, and allowed food and water ad libitum.
Experimental protocols were approved by the Research
Practice Committee at the Hong Kong University of Sci-
ence & Technology.
2.3. Animal Treatment
Female ICR mice (25 - 30 g), with 6 - 10 animals in each
group, were orally treated by gavage with IPP at daily
doses of 0.26 or 0.78 g/kg for 20 doses within 4 weeks
(i.e., 5 doses per week). The low dose is the equivalent of
a human dose (35 mg/kg/day) for IPP. Control animals
received the vehicle (distilled water) only.
2.4. Innate Immunity Assays
2.4.1. Carbon Clearance Test in Vivo―Measurement
of Carbon Clearance
Twenty-four hours after the last dosing with IPP, mice
were injected with 0.2 mL of Indian ink via the tail vein.
Blood samples were withdrawn at 2 and 10 min after in-
jection. An aliquot (20 L) of blood was mixed with 2
mL 0.1% sodium carbonate solution, and the absorbance
of this solution was determined at 600 nm. The rate of
carbon clearance (K) was calculated from the following
equation:
K = (log A10 – log A2)/(t10 – t2)
where A = absorbance at blood collected at the respect-
tive time point, t = time of blood collection (min).
The phagocytic index (  ) was calculated from the fol-
lowing equation [5]:
 = [body weigh/(liver weight + spleen weight)] × K1/3.
2.4.2. Phagocytocytic Activity of Macrophages in Vivo
Measurement of Phagocytic Rate and Index
Twenty-four hours after the last dosing with IPP, mice
were injected with 0.2 mL of 5% starch solution into the
abdominal cavity to stimulate the aggregation and acti-
vation of macrophages. Two hundred microlitre of 20%
(v/v, in 0.9% saline) CRBC suspension were intraperito-
neally injected into mice 24 hours after the starch ad-
ministration. After 30 min, the restrained mice were sac-
rificed by cervical dislocation with metal forceps, and
their abdominal cavity was opened under aseptic condi-
tions to harvest peritoneal macrophages. The peritoneal
cavity was washed with 2 mL phosphate buffered saline
(PBS) and the PBS wash formed the peritoneal fluid.
Then, 1 mL of peritoneal fluid was collected from each
mouse and constituted the macrophage suspension. An
aliquot (200 L) of macrophage suspension was placed
on a clean glass slide. The slides were then incubated at
37˚C for 30 min, and were then rinsed with PBS. Cells
on the slide were fixed with a mixture of acetone and
methanol (1:1, v/v) for 10 min, and then dried, followed
by staining with Wright-Giemsa solution for 6 min and
washing with distilled water. After drying, the cells on
the slide were examined by light microscopy, with the
counting of at least 100 macrophages in different fields.
The phagocytic rate and index of macrophages were cal-
culated as follows [9]:
Phagocytic rate (PR) = (number of macrophage in-
gesting CRBC/number of total macrophage) × 100%
Phagocytic index (PI) = number of total ingested CR-
BC/number of macr oph a ge in gest in g CRBC.
2.4.3. Natural Killer (NK) Cell Activity
2.4.3.1. Isolation of Splenocytes (Effector Cells)
Twenty-four hours after the last dosing with IPP, mice
were sacrificed as described above. Spleens were asep-
ticcally removed from the dissected mice with scissors
and forceps in cold RPMI-1640 medium (without phenol
red). Then, a single cell suspension was obtained by pres-
sing the spleen between the frosted ends of two micro-
scope slides with a gentle circular motion until only the
empty capsule remained. Erythrocytes in the cell mixture
were removed by hypotonic lysis with water. Finally, the
splenocytes were suspended at a final dilution of 1 × 107
cells/mL in RPMI-1640 medium (without phenol red)
supplemented with 5% heat-inactivated fetal calf serum
(HIFBS).

H.-Y. LEUNG ET AL.
180
2.4.3.2. Measurement of NK Cell Activity
YAC-1 cells, which were used as target cells (T), were
seeded in 96-well U-bottom culture plates at 2 × 104
cells/well in RPMI-1640 medium (without phenol red)
supplemented with 5% HIFBS. Spleen cells, prepared as
described above, were used as effector cells (E), and they
were added at 1 × 106 cells/well to give an E/T ratio of
50:1. The cell mixture was then incubated for 24 hours at
37˚C in atmospheric air containing 5% CO2. After incu-
bation, lactate dehydrogenase (LDH) activity in the cul-
ture medium was measured. Briefly, the cell mixture was
centrifuged at 540 × g for 5 min and 100 L of the resul-
tant supernatant was then mixed with 100 L LDH sub-
strate buffer. The reaction mixture was incubated at 37˚C
for 10 min in the dark. The reaction was terminated by
adding 30 L 1M HCl. Absorbance of the reaction mix-
ture was measured at 570 nm. NK cell activity, which was
estimated by the following equation, was expressed as
the percentage of target cells being killed [10]:
NK cell activity (%) = [(Aii-Ai-Aiii)/(Aiv-Ai)] ×100%
where A = absorbance value of the respective experi-
mental sample at 570 nm,
i, denotes basal LDH release from target cells;
ii, denotes LDH release from a mixture of target cells
and effector cells;
iii, basal LDH spontaneously released from effector
cells;
iv, denotes total LDH from target cells.
2.4.4. Tumor Growth Inhibitory Activity Tumor Cell
Inoculation
Adult male ICR mice (~30 g) were randomized into 4
groups, with at least 10 animals in each. Male instead of
female mice were used according to the method of Wang
et al. [11]. An aliquot (200 L) of Sarcoma-180 cells at a
cell concentration of 107 cells/mL was subcutaneously
injected into the right groin of mice on day 1. Twenty-
four hours after the inoculation, mice were treated with
IPP at daily doses of 0.26 and 0.78 g/kg for 20 days until
sacrifice. Body weight and tumor weight were measured,
and data were expressed as tumor weight per g body
weight (i.e. tumor index).
2.5. Adaptive Immunity Assays
2.5.1. Hemolysis of Sheep Red Blood Cell (SRBC) at
50% (HC50)
Female ICR mice (25 - 30 g) were intragastrically treated
with IPP, as described above. In the immunosuppressed
groups, on the day of dose 15, mice were intraperitoneal-
ly administered cyclophosphamide (CYC) at 150 mg/kg.
Measurement of HC50
Aliquots (0.2 mL) of 2% sheep red blood cells (SRBC)
were injected into the abdominal cavity of mice on the
day of dose 18. Twenty-four hours after the last dosing
with IPP, blood samples were drawn and serum samples
were obtained thereafter. Serum samples were diluted at
1:(100 - 400) (v/v) with saline, 200 L of diluted serum
samples were added into glass tubes, and then 100 L of
20% SRBC suspension and 200 L of 6-fold diluted
guinea pig complement were added into the tubes. After
incubation at 37˚C for 30 min, the reaction was stopped
by putting the tubes in an ice bath, and the reaction mix-
tures were centrifuged at 960 × g for 10 min. An aliquot
(200 L) of the resultant supernatant was mixed with
600 L of Doshi reagent (0.2 g potassium ferricyanide,
0.05 g potassium cyanide, 1.0 g sodium hydrogen car-
bonate in 1 L distilled water). In parallel, absorbance for
100% hemolysis was obtained from the following prepa-
ration: 100 L 20% SRBC mixed with 400 L red blood
cell lysis buffer, and then 200 L of the mixture was
added into 600 L Doshi reagent to develop the color for
the measurement of absorbance. The absorbance at 550
nm for assay mixtures was measured and the 50% hemo-
lytic concentration (HC50) of the serum was estimated by
the following equation [12]:
Sample HC50 = (sample absorbance/50% hemolytic
absorbance) × dilution factor.
2.5.2. Con A-Induced Blastogenesis of Mouse
Splenocytes in Vitro and ex Vivo
2.5.2.1. Isolation of Mouse Splenocytes
All procedures were conducted under aseptic conditions.
Splenic tissues were obtained by dissection after sacrifice
as described above from female ICR mice (25 - 30 g),
and isolated splenocytes were resuspended in RPMI-
1640 medium supplemented with 10% HIFBS at a final
concentration of 5 × 106 viable cells/mL. The viability of
isolated splenocytes was determined by Trypan blue ex-
clusion.
2.5.2.2. Ex Vivo Immunomodulatory Activity
Twenty-four hours after the last dosing with IPP, animals
were sacrificed as described above, and splenic tissues
were obtained under aseptic conditions for isolation of
splenocytes, as described above. Mouse splenocytes (106
cells) were cultured in a medium, in the absence or pres-
ence of Con A, in a final volume of 100 L. Con A was
added at final concentrations of 0.5, 1, 2 or 4 g/mL.
Splenocyte index was also estimated by the total number
of splenocytes per gram body weight.
2.5.2.3. In Vitro Immunomodulatory Activity
Isolated mouse splenocytes were cultured in a medium,

181 H.-Y. LEUNG ET AL.
in the presence or absence of Con A, with or without IPP
(dissolved in PBS), in a final volume of 100 L in 96-
well flat bottom microtiter plates. Con A (prepared in
aqueous solution) was added at final concentrations of
0.5, 1, 2 or 4 g/mL. Aliquots of IPP (10 L) were added
at 3 increasing final concentrations in the range 1.56 -
12.5 µg/mL. Control cells received 10 L of vehicle (PBS)
only. Three separate experiments were performed, and
data were expressed as percent change with respective to
the IPP-untreated control.
2.5.2.4. Measurement of Splenocyte Proliferation
Splenocytes were cultured for 72 hours at 37˚C in a hu-
midified atmosphere of air containing 5% CO2. At the
end of the culture period, the extent of splenocyte prolif-
eration was determined by a colorimetric assay using
MTT-based cell proliferation kit I. The extent of Con A-
stimulated proliferation of isolated splenocytes was es-
timated by computing the area under the curve (AUC) of
the graph plotting the net absorbance (mean absorbance
of cells stimulated with Con A―mean absorbance of
cells not stimulated with Con A) against Con A concen-
tration. The effect of IPP on the extent of Con A-stimu-
lated proliferation was compared with that of the IPP-
untreated control, and data were expressed as percent
control [13].
2.5.3. Dinitrofluorobenzene-Induced Delayed-Type
Hypersensitivity Mouse Ear Swelling Assay
Animals were treated with IPP as described above. On
the days of dose 17 and 18, female mice were sensitized
by topical application of 25 L of 0.5% (v/v) DNFB in
acetone-olive oil (4/1, v/v) onto a designated shaved sur-
face of the abdomen. Topical application of 0.2% DNFB
on both ears did not cause a primary reaction in unsensi-
tized mice. The immunosuppression was induced by in-
traperitoneal injection of CYC at 300 mg/kg three days
after the last sensitization. To elicit delayed-type hyper-
sensitivity (DTH), animals were challenged by applying
20 L of 0.2% DNFB in a mixture of acetone and olive
oil (v/v, 4/1) on the dorsal side of both ears two days
after immunosuppression. The ear swelling reaction was
assessed 24, 48 and 72 hours post-challenge by measure-
ing the thickness of the ears with a micrometer. The ex-
tent of edema was estimated for each ear with reference
to the difference in thickness before and after DNFB
challenge. The extent of enhancement in DTH in IPP-
treated animals was estimated by comparison with IPP-
untreated controls [14].
2.5.4. Statistical Analysis
Data were analyzed by one-way ANOVA using SPSS
statistical software. Significant differences between two
groups were determined by Least Significant Difference
when P < 0.05.
3. Results
3.1. Innate Immunity
IPP treatment (0.26 or 0.78 g/kg × 20) increased the pha-
gocytic index in mice (~20% - 22%), as assessed by the
carbon clearance test (Figure 1). IPP treatment signifi-
cantly increased the phagocytic rate of macrophages in
mice, with the maximum degree of stimulation (190%)
being observed at the dose of 0.26 g/kg (Figure 2). How-
ever, no detectable changes in macrophage phagocytic
index were observed.
IPP treatment at a dose of 0.78 g/kg significantly in-
creased (129%) NK cell activity in mice (Figure 3). The
increased NK cell activity resulting from IPP administra-
tion was accompanied by a significant suppression (62%)
of tumor growth in mice (Figure 4).
3.2. Adaptive Immunity
Serum from SRBC-immunized mice showed an enhan-
cement in hemolytic activity on SRBC, as evidenced by
an increase in the degree of dilution of serum that caused
50% hemolysis (Figure 5). The hemolytic activity of the
se- rum was greatly reduced in immunosuppressed mice.
IPP pretreatment (0.26 or 0.78 g/kg × 20) caused a
dose-dependent restoration of hemolytic activity of serum
in SR- BC-immunized and CYC-immunosuppressed mice,
with the maximum stimulation being 56%, when com-
pared with IPP-untreated and immunosuppressed con-
trols.
Figure 1. Effect of long-term IPP treatment on carbon
clearance activity in mice. Animals were treated with In-
finitus Polysac Plus (IPP) administered orally at daily doses
of 0.26 or 0.78 g/kg for 20 doses within 4 weeks. The carbon
clearance test was performed and phagocytic index (α) was
estimated, as described in Materials and methods. Values
given are means ± S.E.M., with n = 6 - 10. *Significantly
different from the IPP-untreated control (P < 0.05).

H.-Y. LEUNG ET AL.
182
(a)
(b)
Figure 2. Effect of long-term IPP treatment on phagocytic
activity of macrophages in mice. Animals were treated with
IPP as described in Figure 1. Phagocytic activity of macro-
phages was measured as described in Materials and meth-
ods, and data were expressed as (a) phagocytic rate and (b)
phagocytic index. Values given are means ± S.E.M., with n
= 6 - 10. *Significantly different from the IPP-untreated
control (P < 0.05).
Figure 3. Effect of long-term IPP treatment on natural kil-
ler cell activity in mice. Animals were treated with IPP as
described in Figure 1. Natural killer cell activity was meas-
ured as described in Materials and methods, and data were
expressed as percent natural killer cell activity. Values
given are means ± S.E. M., with n = 6 - 10. *Significantly
different from the IPP-untreated control (P < 0.05).
IPP treatment significantly increased the splenocyte
index of mice (20%) at a dose of 0.76 g/kg (Figure 6).
While IPP pretreatment did not produce any detectable
changes in the extent of Con A-stimulated proliferation
of mouse splenocytes ex vivo (Data not shown), it dose-
dependently (1.56-12.6 µg/mL) potentiated (3-14%) the
Con A-stimulated proliferation of mouse splenocytes in
vitro (Figure 7).
DNFB induced DTH in sensitized mice, as evidenced
by the increase in ear thickness, with the maximum in-
crease in the extent of edema (35 fold) occurring at 48 h
post-DNFB challenge (Figure 8). DNFB-induced DTH
was almost completely inhibited in immunosuppressed
mice. IPP pretreatment at a dose of 0.78 g/kg partially
but significantly restored the DNFB-induced DTH reac-
tion in immunosuppressed mice, with the maximum in-
crease in the extent of edema formation (10 fold) occur-
ring at 24 h post-DNFB challenge at a dose of 0.78 g/kg.
Figure 4. Effect of long-term IPP treatment on tumor grow-
th induced by Sarcoma-180 cell inoculation in mice. Ani-
mals were inoculated with Sarcoma-180 cells and then
orally treated with IPP at daily doses of 0.26 or 0.78 g/kg
for 20 doses. Body and tumor weight were measured, and
data were expressed as tumor index (i.e., tumor weight per
g body weight). Values given are means ± S.E.M., with n =
10. *Significantly different from the IPP-untreated control
(P < 0.05).
Figure 5. Effect of long-term IPP treatment on the concen-
tration of serum required to produce 50% hemolysis (HC50)
of sheep red blood cells in (SRBC)-immunized and immu-
nosuppressed mice. Animals were treated with IPP and
then immunized with sheep red blood cells (SRBC) as de-
scribed in Materials and Methods. Cycophosphamide (CYC)
was administered intraperitoneally at 150 mg/kg. Hemolytic
activity of serum samples was measured, and data were
expressed as HC50. Values given are means ± S.E.M., with n
= 6 - 10. *Significantly different from the CYC control
group (P < 0.05).

183 H.-Y. LEUNG ET AL.
Figure 6. Effect of long-term IPP treatment on splenocyte
index in mice. Animals were treated with IPP as described
in Figure 1. The total number of splenocytes was measured,
and data were expressed as splenocyte index (total number
of cells/g body weight). Values given are means ± S.E.M.,
with n = 6. *Significantly different from the IPP-untreated
control (P < 0.05).
Figure 7. Effect of long-term IPP co-incubation on Con
A-induced blastogenesis in mouse splenocytes in vitro. Iso-
lated mouse splenocytes were subjected to Con A chal-
lenge (1 - 4 µg/mL) in the absence or presence of IPP (1.56 -
12.5 µg/mL). The extent of Con A-stimulated blastogensis
was estimated, and data were expressed as percent control
(compared to the Con A-stimulated group without IPP
co-incubation). Values given are means ± S.E.M., with data
obtained from 3 separate experiments. *Significantly dif-
ferent from the non-IPP co-incubated control (P < 0.05).
4. Discussion
Long-term IPP treatment at oral doses (which didn’t show
any observable adverse effect in the fed mice), including
an equivalent of a human dose, was found to enhance
phagocytic activity in mice, as evidenced by increases in
carbon clearance and phagocytic rate of macrophages.
The increased rate of carbon clearance, indicative of en-
hanced Kupffer cell phagocytosis [15], is consistent with
the activation of macrophages in IPP-treated mice. A
recent study in chickens showed that the extent of pha-
gocytosis by monocytes/macrophages in vitro corre-
lated with the capacity in pathogen clearance and anti-
body production in vivo [16]. IPP treatment also stimu-
lated NK cell activity in mice. NK cells play an impor-
tant role in serving as the first line of defense against
Figure 8. Effect of long-term IPP treatment on dinitrofluo-
robenzene (DNFB)-induced delayed-type hypersensitivity in
immunosuppressed mice. Animals were treated with IPP
and then sensitized with dintrofluorobenzene (DNFB) as
described in Materials and methods. CYC was adminis-
tered intraperitoneally at 300 mg/kg. Then animals were
challenged by topically applying DNFB on the dorsal side of
both ears, and the ear thickness was measured at 24, 48 and
72 hours post-DBFB challenge (first bar, second bar and
third bar, respectively, shown in the figure). Data were ex-
pressed as differences in ear thickness before and after
DNFB challenge. Values given are means ± S.E.M., with n =
10. A, control group without sensitization and challenge; B,
control group without sensitization but with challenge; C,
control group with sensitization and challenge; D, CYC-
treated group with sensitization and challenge; E, CYC
group with IPP treatment (0.26 g/kg) followed by sensitiza-
tion and challenge; F, CYC group with IPP treatment (0.78
g/kg) followed by sensitization and challenge. *Signifi-
cantly different from the CYC-treated and IPP-untreated
control group (P < 0.05).
microbial infections as well as providing surveillance on
neoplastic cells [17]. In this connection, the stimulation
of NK cell activity by IPP treatment was associated with
the suppression of tumor growth in Sarcoma 180 cell-
inoculated mice. Conceivably, IPP treatment can stimu-
late the innate (i.e. non-specific) immune system, pre-
sumably via activation of macrophage and NK cells, and
hence increase the resistance to microbial infection and
growth of neoplastic cells in the body.
The adaptive (i.e., specific) immune system is respon-
sible for the production of antigen-specific antibodies
that protect against infectious agents by neutralizing vi-
ruses, destroying targets by antibody-dependent cellular
cytotoxicity and complement-mediated cell lysis, etc.
[18,19]. In this regard, IPP treatment was found to re-
store the hemolytic activity on SRBC of serum in SRBC-
sensitized and CYC-immunosuppressed mice, indicative
of a potentiation of adaptive humoral immunity [6]. The
generation of an adaptive humoral immune response in-
volves the functional interaction between T- and B-lym-
phocytes such that the antigen-activated T helper cells
produce a variety of cytokines (such as interleukin-2, in-
terleukin-6 and interferon-
 ) that then cause B-lympho-
cytes to proliferate and differentiate into antibody-pro-

H.-Y. LEUNG ET AL.
184
ducing plasma cells [20,21]. In this connection, IPP
treatment produced an increase the splenocyte index in
mice, and IPP co-incubation also potentiated Con A-sti-
mulated splenocyte blastogenesis in vitro. Given that
Con A-stimulated splenocyte blastogenesis is a measure
of T-lymphocyte immunity [22], the ability of IPP to
enhance Con A-stimulated splenocyte proliferation is
consistent with an enhanced humoral immune response
produced by IPP treatment. DTH reactions, such as
chemical contact allergy as was the case for DNFB, are
regarded as adaptive cell-mediated immune responses
involving T helper-1 type cells [23]. IPP treatment was
found to restore the DNFB-induced increase in ear
thickness in DNFB-sensitized and CYC-immunosup-
pressed mice, indicative of potentiation of adaptive cell-
mediated immunity [24]. The mixture of herb-derived
polysaccharides present in IPP produces immunostimu-
latory effects on both innate and adaptive immune sys-
tems in mice. Supplementation with polysaccharides de-
rived from various edible fungi has previously been shown
to increase the phagocytic activity of macrophages and
NK cell activity in mice [2]. However, no detectable ef-
fects were produced by edible fungus polysaccharides on
the hemolytic activity on SRBC and DNFB–induced
DTH in mice.
In conclusion, long-term IPP treatment enhanced both
innate and adaptive immunity in mice. The immunopha-
rmacological profile of IPP provides a scientific rationale
for the time-honored use of this compound polysaccha-
ride-containing herbal health product (which is generally
regarded as safe according to the high usage in Mainland
China) for promoting health.
5. Acknowledgements
This work was supported by Lee Kum Kee Health Prod-
ucts Group Ltd., Hong Kong, China.
6. References
[1] S. P. Wasser, “Medicinal Mushrooms as a Source of An-
titumor and Immunomodulating Polysaccharides,” Ap-
plied Microbiology and Biotechnology, Vol. 60, No. 3,
2002, pp. 258-274. doi:10.1007/s00253-002-1076-7
[2] Y. Yin, W. Fu, M. Fu, G. He and L. Traore, “The Im-
mune Effects of Edible Fungus Polysaccharides Com-
pounds in Mice,” Asia Pacific Journal of Clinical Nutri-
tion, Vol. 16, 2007, pp. 258-260.
[3] B. Bin-Hafeez, R. Haque, S. Parvez, S. Pandey, I. Sayeed
and S. Raisuddin, “Immunomodulatory Effects of Fenu-
Greek (Trigonella foenum graecum L.) Extract in Mice,”
International Immunopharmacology, Vol. 3, No. 2, 2003,
pp. 257-265. doi:10.1016/S1567-5769(02)00292-8
[4] X. T. Chen, J. Li, H. L. Wang, W. M. Cheng, L. Zhang
and J. F. Ge, “Immunomodulating Effects of Fractioned
Polysaccharides Isolated from Yu-Ping-Feng-Powder in
cyclophosphamide-Treated Mice,” The American Journal
of Chinese Medicine, Vol. 34, No. 4, 2006, pp. 631-641.
doi:10.1142/S0192415X06004168
[5] X. Yang, D. Guo, J. Zhang and M. Wu, “Characterization
and Antitumor Activity of Pollen Polysaccharide,” Inter-
national Immunopharmacology, Vol. 7, No. 4, 2007, pp.
427-434. doi:10.1016/j.intimp.2006.10.003
[6] G. S. Ladics, “Use of SRBC Antibody Responses for
Immunotoxicity Testing,” Meth ods, Vol. 41, No. 1, 2007,
pp. 9-19. doi:10.1016/j.ymeth.2006.07.020
[7] J. Parkin and B. Cohen, “An Overview of the Immune
System,” Lancet, Vol. 357, No. 9270, 2001, pp. 1777-
1789. doi:10.1016/S0140-6736(00)04904-7
[8] M. Dubois, K. A. Gilles, J. K. Hamilton, P. A. Rebers and
F. Smith, “Colorimetric Method for Determination of
Sugars and Related Substances,” Analytical Chemistry,
Vol. 28, 1956, pp. 350-356.
doi:10.1021/ac60111a017
[9] T. Wang, F. Fu, L. Zhang, B. Han, M. Zhu and X. Zhang,
“Effects of Escin on Acute Inflammation and the Immune Sys-
tem in Mice,” Pharmacological Reports, Vol. 61, 2009, pp.
697-704.
[10] G. C. Huang, L. S. Wu, L. G. Chen, L. L. Yang and C. C.
Wang, “Immuno-Enhancement Effects of Huang Qi Liu
Yi Tang in a Murine Model of Cyclophosphamide-In-
duced Leucopenia,” Journal of Ethnopharmacology, Vol.
109, No. 2, 2007, pp. 229-235.
doi:10.1016/j.jep.2006.07.023
[11] W. Wang, Q. L. Guo, Q. D. You, K. Zhang, Y. Yang, J.
Yu, W. Liu, L. Zhao, H. Y. Gu, Y. Hu, Z. Tan and X. T.
Wang, “The Anticancer Activities of Wogonin in Murine
Sarcoma S180 Both in vitro and ex vivo,” Biological &
Pharmaceutical Bulletin, Vol. 29, No. 6, 2006, pp. 1132-
1137. doi:10.1248/bpb.29.1132
[12] X. M. Wen, Y. L. Zhang, X. M. Liu, S. X. Guo and H.
Wang, “Immune Responses in Mice to Arecoline Medi-
ated by Lymphocyte Muscarinic Acetylcholine Recep-
tor,” Cell Biology International, Vol. 30, 2006, pp. 1048-
1053. doi:10.1016/j.cellbi.2006.09.015
[13] K. M. Siu, D. H. Mak, P. Y. Chiu, M. K. Poon, Y. Du and
K. M. Ko, “Pharmacological Basis of ‘Yin-Nourishing’
and ‘Yang-Invigorating’ Actions of Cordyceps, a Chinese
Tonifying Herb,” Life Sciences, Vol. 76, No. 4, 2004, pp.
385-395. doi:10.1016/j.lfs.2004.07.014
[14] L. Quemeneur, M. C. Michallet, C. Ferraro-Peyret, P.
Saint-Mezard, J. Benetiere, M. T. Ducluzeau, J. F. Nico-
las and J. P. Revillard, “Immunosuppressive Antimetabo-
lites Inhibit Induction of Contact Hypersensitivity While
Lymphoablative Drugs Also Prevent Its Expression,”
European Journal of Dermatology, Vol. 13, 2003, pp.
540-547.
[15] K. Yamanouchi, H. Zhou, N. Roy-Chowdhury, F.
Macaluso, L. Liu, T. Yamamoto, G. R. Yannam, C. Enke,
T. D. Solberg, A. B. Adelson, J. L. Platt, I. J. Fox, J.
Roy-Chowdhury and C. Guha, “Hepatic Irradiation Aug-

H.-Y. LEUNG ET AL.
185
ments Engraftment of Donor Cells Following Hepatocyte
Transplantation,” Hepatology, Vol. 49, No. 1, 2009, pp.
258-267. doi:10.1002/hep.22573
[16] S. F. Sun, Q. Z. Pan, X. Hui, B. L. Zhang, H. M. Wu, H.
Li, W. Xu, Q. Zhang, J. Y. Li, X. M. Deng, J. W. Chen, Z.
X. Lian and N. Li, “Stronger in vitro Phagocytosis by
Monocytes-Macrophages Is Indicative of Greater Patho-
gen Clearance and Antibody Levels in vivo,” Poultry
Science, Vol. 87, No. 9, 2008, pp. 1725-1733.
doi:10.3382/ps.2007-00202
[17] J. Wang, H. Yu, X. Shen, Z. Long, S. Yan, K. Nenkei, A.
Hideo and Y. Li, “Effect of Chinese Soft-Shell Turtle
Egg Powder on Immune Functions in Mice,” Food and
Agricultural Immunology, Vol. 15, No. 3-4, 2003, pp.
207-216. doi:10.1080/09540100400014557
[18] A. F. Ochsenbein and R. M. Zinkernagel, “Natural Anti-
bodies and Complement Link Innate and Acquired Im-
munity,” Immunology Today, Vol. 21, No. 12, 2000, pp.
624-630.
doi:10.1016/S0167-5699(00)01754-0
[19] T. Storni, T. M. Kündig, G. Senti and P. Johansen, “Im-
munity in Response to Particulate Antigen-Delivery Sys-
tems,” Advanced Drug Delivery Reviews, Vol. 57, No. 3,
2005, pp. 333-355. doi:10.1016/j.addr.2004.09.008
[20] R. König and W. Zhou, “Signal Transduction in T Helper
Cells: CD4 Coreceptors Exert Complex Regulatory Ef-
fects on T Cell Activation and Function,” Current Issues
in Molecular Biology, Vol. 6, 2004, pp. 1-15.
[21] M. McHeyzer-Williams, L. McHeyzer-Williams, J. Panus,
R. Pogue-Caley, G. Bikah, D. Driver and M. Eisenbraun,
“Helper T-Cell-Regulated B-Cell Immunity,” Microbes
and Infection, Vol. 5, No. 3, 2003, pp. 205-212.
doi:10.1016/S1286-4579(03)00012-1
[22] F. Cerqueira, A. Cordeiro-Da-Silva, C. Gaspar-Marques,
F. Simoes, M. M. Pinto and M. S. Nascimento, “Effect of
Abietane Diterpenes from Plectranthus Grandidentatus on
T- and B-Lymphocyte Proliferation,” Bioorganic & Me-
dicinal Chemistry, Vol. 12, No. 1, 2004, pp. 217-223.
doi:10.1016/j.bmc.2003.10.006
[23] R. J. Dearman and I. Kimber, “Role of CD4(+) T Helper
2-Type Cells in Cutaneous Inflammatory Responses In-
duced by Fluorescein Isothiocyanate,” Immunology, Vol.
101, 2000, pp. 442-451.
doi:10.1046/j.1365-2567.2000.01126.x
[24] R. J. Dearman, C. J. Betts, N. Humphreys, B. F. Flanagan,
N. J. Gilmour, D. A. Basketter and I. Kimber, “Chemical
Allergy: Considerations for the Practical Application of
Cytokine Profiling,” The Journal of Toxicological Sci-
ences, Vol. 71, No. 2, 2003, pp. 137-145.
doi:10.1093/toxsci/71.2.137