International Immunopharmacology (2008) 8, 4350

w w w. e l s e v i e r. c o m / l o c a t e / i n t i m p

Macrophage immunomodulatory activity of

polysaccharides isolated from Glycyrrhiza uralensis fish
Anwei Cheng a , Fachun Wan b , Jiaqi Wang c , Zhengyu Jin a,, Xueming Xu a
a
School of Food Science and Technology, Jiangnan University, Wuxi 214122, China
b
Institute the Animal Science and Veterinary Medicine, Shandong Academy of Agriculture Science, Jinan 250100, China
c
Institute the Animal Science, Chinese Academy of Agriculture Science, Beijing 100094, China

Received 8 July 2007; received in revised form 3 October 2007; accepted 4 October 2007

KEYWORDS Abstract
Glycyrrhiza uralensis Fish;
Polysaccharides; The objective of this study was to evaluate the immunomodulatory effects of the purified glycyrrhiza
Immunomodulator; polysaccharides (GP) on the activity of macrophages. A purified fraction of water-soluble
Macrophage; polysaccharides, with estimated molecular weight of 10 kDa, was isolated from Glycyrrhiza uralensis
Cytokine Fish using ion exchange and size exclusion chromatography. The results indicate that GP increased the
pinocytic activity, the production of nitric oxide (NO), interleukin-1 (IL-1), IL-6 and IL-12 in a dose-
dependent manner. The production of IL-1 was induced by GP at a dose of 10 g/mL; but, NO, IL-6 and
IL-12 was significantly induced at 100 g/mL. A time-dependent enhancement showed that the
production of IL-1, NO and IL-12 were significantly increased within 6 h. Superoxide anion (O2 )
production by macrophages from GP-treated mice was higher than that of cells from untreated mice.
Moreover, cells from both untreated and treated mice responded to phorbol 12-myristate 13-acetate
(PMA) treatment; however, the O2 production was higher in the cells from treated mice than that of
cells from untreated mice. Our data suggest that the beneficial therapeutic effects of GP may be
attributed partly to its ability to modulate macrophage immune functions.
2007 Published by Elsevier B.V.

1. Introduction lipopolysaccharides (LPS), and several inflammatory cyto-

Macrophages occupy a unique niche in the immune system, in
that they not only can initiate innate immune responses but
they can also be effector cells that contribute to the resolution
of these responses [1], such as fighting infection, inflamma-
tion, angiogenesis and in the promotion of wound healing.
Macrophages can be stimulated by bacterial products including
Corresponding author. Tel.: +86 510 85913299; fax: +86 510
85811950.
E-mail address: jinlab2008@yahoo.com.cn (Z. Jin).
kines, interleukin-1 (IL-1), IL-6, IL-12 and nitric oxide (NO)
released by muramyl dipeptide. Macrophages kill tumor cells
and damage tissues during inflammation by two separate
oxidative pathways involving the synthesis of superoxide anion
(O2 ) and NO [2], which are formed, respectively, by NADPH
oxidase and nitric oxide synthases (iNOS) [3,4]. These cells are
also able to produce a variety of cytokines, such as interleukin
(IL), interferon (IFN), tumor necrosis factor (TNF) and active
substances, such as prostaglandins [5].
Recently, substances for enhancing host defense responses
were isolated from microorganisms, fungi [6] and plants
[7,8]. Plant-derived polysaccharides are the best known and

1567-5769/$ - see front matter 2007 Published by Elsevier B.V.

doi:10.1016/j.intimp.2007.10.006
44
most potent immunomodulatory substances [7] and have
been shown to be clinically therapeutic [9]. Their chemical
properties and biological activities have been studied
extensively. Plant polysaccharides have been shown to
exhibit anti-inflammatory [10], anti-hypoglycemic [11],
anti-bacterial [12], and anti-tumor [13], and anti- comple-
mentary activities. Indeed, the basic mechanism of the
immuno-stimulatory, anti-tumor, bactericidal and other
therapeutic effects of botanical polysaccharides are thought
to occur via macrophage stimulation and modulation of the
complement system [14,15]. Furthermore, modulation of
these systems can significantly impact both humoral and
cellular immune responses [16].
Glycyrrhiza uralensis Fish is a well-known Chinese herbal
medicine used for the treatment of various diseases as well
as a tonic medicine for thousands of years. This herb has long
been valued as a demulcent, to relieve respiratory ailments
(such as allergies, bronchitis, cold, sore throats and
tuberculosis), stomach burn including heart burn, gastritis,
inflammatory disorders, skin diseases and liver problems
[17]. Glycyrrhiza contains a variety of substances. The water
extract of glycyrrhiza has been widely used in medicine,
pharmacology and food industry because of its physical and
functional properties. The medicinal and pharmacological
uses of glycyrrhiza have been described in several studies
[1820]. Glycyrrhiza polysaccharide (GP), one of the main
active ingredients of glycyrrhiza is attributed to many
healing properties of the herb. Recently, it has been reported
that GP has many functions such as immunity regulation [21],
phagocytosis [22], anti-complement [23], anti-virus [24],
anti-tumor [25], and it has low cellular toxicity [24].
The aqueous extracts from glycyrrhiza and the structure of
their oligo and polysaccharides have been described
[23,24,26]. Studies indicated that GP at least includes three
core structures [27]: firstly, a backbone chain composed of
beta-1,3-linked D-galactose residues, three-fifths of the
galactose units in the backbone carry side chains composed of
beta-1,3- and beta-1,6-linked D-galactosyl residues at position
6 [23]; secondly, glycosidic units in the backbone carry side
chains composed of mainly alpha-1,5-linked L-arabino-beta-1,6-
or 1,3-linked D-galactose residues at position 6 [26]; thirdly,
glycosidic units were composed of (1 4) linked D-glucan [24].
The objective of this study was to demonstrate that the
immunomodulatory effects of GP on mice macrophages
associated with nitric oxide, reactive oxygen species, and
cytokine releases. The effects of GP to modulate macro-
phage function suggest it may contribute to the therapeutic
potential of aqueous extract derived from this herb.
2. Materials and methods
2.1. Plant material
Roots of Glycyrrhiza uralensis Fish were collected from Inner
Mongolia Municipality, China, and identified by Dr. Houwei Wang,
Shandong University of Traditional Chinese Medicine, China. A
vouchers specimen was deposited at the School of Food Science
and Technology, JiangnanUniversity, China.
2.2. Preparation of glycyrrhiza extract
Briefly, aqueous extracts of glycyrrhiza roots were obtained at
9095 C and at a water/root mass ratio of 10:1 (v/m) for 34 h.
A. Cheng et al.
After filtration to remove debris fragments, the filtrate was
concentrated in a rotary evaporator. Proteins were removed by
adding 15% (m/m) of trichloroactic acid, the filtrate was
neutralized by 1 mol/L NaOH, and then centrifuged to remove
the precipitates. Subsequently, three volumes of ethanol were
added to the supernates, and the precipitates were collected
by centrifugation and washed with acetone for three times. The
precipitates were dissolved in distilled water and then applied
to a DEAE- cellulose column (2.5 cm 60 cm). The bound
materials were eluted with a linear gradient of NaCl (0
2 mol/L). Total polysaccharide obtained from the elution was
fractionated by chromatography on a Sephacry S-400 HR column
(3.5 cm 100 cm) (Pharmacia, Sweden) and eluted with distilled
water. The main fraction of GP was then dialyzed and
lyophilized. The dried extract was dissolved in phosphate
buffer solution (PBS) and filtered through 0.22 m filter before
use.
2.3. Materials
Phorbol 12-myristate 13-acetate (PMA), ferricytochrome C,
lipopolysaccharide (LPS) and polymyxin B (PB) were purchased
from Sigma (St. Louis, USA). RPMI-1640 medium was from Gibco
(NY, USA). Bovine calf serum was from Hyclone (Utah, USA).
ELISA Kits for IL-1, IL-6 and IL-12 were from Jingmei (Shenzhen,
China) Co. Ltd.
2.4. Animals
Male BALB/c mice between 68 weeks old (weight: 18.6 1.0 g)
were purchased from the Experimental Animal Center of Peking
University, China. Mice were randomized and transferred to
assigned cages containing sawdust bedding (three mice per
cage), food and water were provided ad libitum. Mice holding
rooms were kept at 2125 C and 50% relative humidity with a
12 h/12 h light/dark cycle. Soluble starch was prepared by
dissolving 5 g in 100 mL distilled water, and the solution was
filtered and stored at 4 C before using. The diluents of the
solution (5 g/100 mL) was injected intraperitoneally to mice
with the dose of 1 mL/mouse and then bred for 3 d.
2.5. Isolation of peritoneal macrophages
Peritoneal fluid from male BALB/c mice were harvested from
peritoneal cavities by infusing 10 mL ice-cold sterile PBS (pH
7.27.4). After centrifugation at 1000 rpm/min for 5 min, the
cell pellets were suspended in RPMI-1640 supplemented with
10% (v/v) bovine calf serum, penicillin 100 U/mL, and
streptomycin 100 U/mL and seeded in 96-well plate at a cell
density of 5 105 cells/mL, and allowed to adhere for 3 h at
37 C in 5% CO 2 humidified incubator. After 3 h incubation, non-
adherent cells were removed by washing twice with PBS and
freshly prepared medium was added [28]. The viability of the
adherent cells was assessed by trypan blue exclusion test, and
the proportion of macrophages was determined by cell
morphology under a microscope.
2.6. Pinocytic activity assay
Peritoneal macrophages were suspended in RPMI-1640 contain-
ing various concentrations of GP (10, 100, 200, 400 g/mL).
Cells were placed in a 96-well plate and cultured at 37 C, 5%
CO 2 for 72 h. Culture media were removed and 100 L/well of
0.1% neutral red was added, and incubated for 4 h. Media were
discarded, and macrophages were washed for twice with PBS
(pH 7.27.4). Washed Macrophages were resuspended in
Macrophage immunomodulatory activity of polysaccharides isolated from Glycyrrhiza uralensis fish
Figure 1 Gel permeation of extracted polysaccharides by
chromatography using a column of Sephacry S-400 HR (Column
dimension: 3.5 cm 100 cm, flow rate = 0.5 mL/min, fraction
size = 5 mL). Carbohydrate was estimated from each fraction by
phenol sulfuric acid method. Fractions 11 to 31 were pooled as GP.
100 L/well of cell lysing solution and cultured for 2 h. The
absorbance (A 540 nm) was measured using an ELISA reader.
2.7. NO production
Adherent macrophages (5 105 cells/well) were placed in a 96-well
plate and incubated in complete RPMI medium alone or medium
containing various concentrations of GP or LPS (2 g/mL) as a
positive control for 48 h. Nitrite in the culture medium was
determined by Griess reaction [29]. At the end of the culture
period, a total 100 L/well of cell culture medium was incubated
with equal volume of Griess solution (1% sulfanilamide, 0.1% napthyl
ethyl diamine dihydrochloride in 5% phosphoric acid) at room
temperature for 10 min. The absorbance was read at 540 nm, and
the concentrations of NO2 were determined from a least squares
linear regression analysis of a sodium nitrite standard curve.
2.8. Measurement of O2 production
Study for O2 production was conducted using groups of nine mice per
treatment. All control groups received sterile PBS (pH 7.27.4). Mice
were treated intraperitoneally with GP solution. Seven days later,
macrophages were collected and plated (5 105 cells/well) on 24-well
plates. Adherent macrophages were collected as described previously.
For O2 measurement, adherent macrophages (5 105 cells/mL) were
incubated in HBSS (Hank's balanced salt solution) standard reaction
mixture consisting of ferricytochrome C (80 M) in the presence or
absence of PMA (1 g/mL). Absorbance was measured at 540 nm and
the extinction molar coefficient = 2.1 104 m 1 cm 1 was used to
determine reduced cytochrome C [28,30], and results are expressed as
nmol O2 produced per mg cell protein.
2.9. Enzyme linked immunosorbent assay for IL-1, IL-6
and IL-12
Macrophages (5 105 cells/mL) were cultured in the presence of
various concentrations of GP in a 96-well plate in a total volume of
100 L up to 48 h, or cultured for different time in the presence of GP
(100 g/mL). Productions of IL-1, IL-6 and IL-12 were measured using
commercially available ELISA kits.
2.10. Determination of endotoxin contamination
Briefly, 1 mL of Affi-Prep Polymyxin Matrix was packed in a Bio-spin
column, centrifuged at 200 rpm/mim for 2 min, and then 0.5 mL of
GP (100 g/mL), LPS (2 g/mL) or GP (100 g/mL) and LPS (2 g/mL)
45
mixture was added. After incubating overnight at 4 C, the effluent
was recovered from the column by centrifugation at the same
condition [31].
2.11. Statistical analysis
Biological assays were performed in triplicate. Results were
expressed as mean standard deviations (S.D.). Data were analyzed
using Analysis of Variance (SAS 9.0) and T-test to determine the
statistical significance (P 0.01).
3. Results
3.1. Isolation of water-soluble polysaccharide from
glycyrrhiza
Crude polysaccharides were isolated from glycyrrhiza and the
yield of crude polysaccharide was about 4.25%, and the yield of
purified polysaccharide was approximately 0.87%. The crude
polysaccharides were purified by gel permeation using Sephacry
S-400 and Fig. 1 represents the gel permeation profile of ion
exchange-purified polysaccharides. A single peak was found
according to the carbohydrate concentration; fractions 11 to 31
were pooled as purified glycyrrhiza polysaccharides. Compara-
tive analysis of pullulan standards measured by HPLC resulted in
a calibration curve that was linear from molecular weight (MW)
(Log mol.wt = 1.44e + 001 5.53e001T^1, where T is retention
time), with a correlation coefficient is 0.982196. Using this
calibration, the molecular weight of purified GP was approxi-
mately 10 kDa.
3.2. Effects of GP on pinocytic activity
One of the most distinguished features of macrophage activation
would be an increase in pinocytic activity. Pinocytic activity of
GP-activated macrophages was examined by the uptake of
neutral red (0.1%). The results, (Fig. 2), show that a dose-
dependent enhancement of pinocytic activity was observed in
macrophages treated with 10400 g/mL doses of GP. GP
appeared to prime macrophages for an enhanced pinocytic
Figure 2 Effects of GP on pinocytic activity of macrophages.
Cells were placed in a 96-well plate and cultured at 37 C, 5%
CO2 for 72 h. Culture media were removed and 100 L/well of
0.1% neutral red added, and incubated for 4 h. Media were
discarded, and macrophages were washed for three times with
PBS (pH 7.27.4).Washed macrophages were resuspended in
100 L/well of cell lysing solution and cultured for 2 h. The
pinocytic level was measured as the absorbance at 540 nm.
Values are means S.D., (n = 3), P 0.01 vs. control.
46 A. Cheng et al.

recognized as a co-signal [32,33], these experiments were

performed either in the absence or the presence of PMA.
Fig. 4A shows that O2 production by macrophages from mice
inoculated with 0400 mg/kg of GP was 1837 nmol/mg cell
protein in the presence of PMA, whereas, only was 719 nmol/
mg cell protein in the absence of PMA. Fig. 4B shows that
macrophages from GP-treated animals were activated as O2
production after 60 min was 150% greater (P 0.01) than
macrophages from untreated mice. With PMA, cells from
untreated mice increased their O2 production as expected
and it can be observed that macrophages from treated mice
also responded to PMA, but the effect after 60 min was only
40% higher than that of the control (untreated mice).

3.5. GP induced IL-1 in a dose- and time-dependent manner

The effect of GP on the production of IL-1 was dose-dependent

(Fig. 5A). The production of IL-1 was enhanced by GP (10, 100,
200, 400 g/mL). Furthermore, the concentrations of IL-1
production at 100400 g/mL treatments of GP were compar-
able to that elicited by LPS (2 g/mL). A time-dependent

Figure 3 Effects of GP on the production of NO by peritoneal

macrophages in vitro. (A) Macrophages of mice was stimulated
with GP (0, 10, 100, 200 and 400 g/mL) or LPS (2 g/mL) for
48 h. (B) Peritoneal macrophages were incubated with GP
(100 g/mL) for 072 h. The isolated supernatants were mixed
with an equal volume of Griess reagent, nitric production was
measured by an O.D. reading at 540 nm. Each value represents
the mean S.D., (n = 3). P 0.01 vs. control.

activity, which was comparable to or greater than that observed

for LPS, a positive control.

3.3. GP induced NO in a dose- and time-dependent

manner

The experiment investigated whether NO production was

increased in the macrophages of mice stimulated with GP or
LPS for 48 h. NO was detected at a concentration above the
control. As shown in Fig. 3A, a minimum amount of NO was
released when macrophages were exposed to medium alone,
whereas incubation of these cells with increasing amounts of GP
was associated with a dose-dependent increase in NO produc-
tion. Not much NO was produced in the 10 g/mL GP group, but
much higher NO was produced with 100 g/mL GP. Furthermore,
the levels of NO production at 100400 g/mL concentrations of
GP were comparable to or even greater than that elicited by
2 g/mL LPS. To further investigate whether the production of Figure 4 Effects of in vivo GP on the production of O2 by
NO by GP was time-dependent, macrophages were cultured for macrophages. (A) Mice were injected with GP (10, 100, 200 and
672 h at a concentration of 100 g/mL GP. As shown in Fig. 3B, 400 g/mL). Adherent macrophages were collected seven days
at time as early as 6 h, the level of NO induced by GP was higher later, and incubated in a standard reaction mixture consisting
(P 0.01) than control medium alone and continued to increase HBSS, ferricytochrome C (80 M) in the presence or absence of
up to the 72 h. PMA (1 g/mL) for 60 min. Absorbance was measured at 540 nm.
(B) Mice were injected with 200 g/mL GP. Adherent macro-
3.4. Effects of GP on O2 production phages were collected seven days later, and incubated with the
same standard reaction mixture for 090 min. Results are
The effect of GP on O2 production by peritoneal macrophages expressed as nmol O2 /mg cell protein. Each value represents the
is shown in Fig. 4. In the induction of O2 system, PMA is mean S.D., (n = 3). P 0.01 vs. control.
Macrophage immunomodulatory activity of polysaccharides isolated from Glycyrrhiza uralensis fish
Figure 5 Effects of GP on IL-1 secretion of mice macrophages
in vitro. (A) Peritoneal adherent macrophages were incubated
for 72 h in RMPI-1640 medium containing various concentrations
of GP or LPS. (B) Macrophages were isolated from BALB/c mice
(5 105 cells/mL), and cultured in 96-well flat-bottomed
microtiter plate without or with GP (100 g/mL) for 072 h.
Each value represents the mean S.D., (n = 3). P 0.01 vs.
control.
enhancement of IL-1 production was observed in macrophages
treated for 072 h (Fig. 5B). The level of IL-1 induced by GP
(100 g/mL) was higher (P 0.01) than that of the control, and
continued to increase IL-1 production after 72 h incubation.
3.6. GP induced IL-6 in a dose- and time-dependent manner
To assess the effect of GP on the secretion of IL-6, macrophages
were treated with various concentrations of GP up to 72 h. The
secretion of IL-6 is also GP dose-dependent (Fig. 6A). GP (100
400 g/mL) treated groups had an increase (P 0.01) in IL-6
production. Interestingly, GP-activated macrophages produced
large amount of IL-6, reaching approximately 1600 pg/mL, at
400 g/mL GP. This effect of GP was similar to that of LPS. A
time-dependent enhancement of IL-6 production was also
observed in macrophages treated for 072 h (Fig. 6B).
3.7. GP induced IL-12 in a dose- and time-dependent
manner
To examine whether GP-activated macrophages produced
cytokines, the amounts of IL-12 were measured by ELISA. As
shown in Fig. 7A, IL-12 productions were induced by GP (10, 100,
200, 400 g/mL) in a dose-dependent manner, with 1.4-, 1.6-,
2.0-, 3.1-fold induction as compared to the control. The amount
of IL-12 produced in response to 400 g/mL of GP was up to that
47
of LPS (2 g/mL). As shown in Fig. 7B, IL-12 induction by GP is
also time-dependent. However, there was no further increase
beyond 48 h at the dose of 100 g/mL GP.
3.8. Effect of polymyxin B on NO production
To ensure that the effects of GP were not due to endotoxin
contamination, GP was treated with polymyxin B, an inhibitor of
LPS activity, and the immunomodulatory activity was examined
in peritoneal macrophages. As shown in Fig. 8, passage of LPS
solution (2 g/mL) through polymyxin B-affinity column reduced
the nitric oxide-inducing activity almost completely, indicating
that polymyxin B-affinity column absorbed LPS almost comple-
tely. Passage of GP solution (100 g/mL) with polymyxin B-
affinity column, however, did not reduce the nitric oxide-
inducing activity to a significant level. To exclude possible
interference of the complex-forming activity of polymyxin B by
unknown substances contained in the GP solution, GP was mixed
with LPS, passed through polymyxin B-affinity column, and then
the nitric oxide inducing activity in macrophages was measured.
As shown in Fig. 8, polymyxin B-affinity column could remove
LPS from the mixture of GP and LPS. These results indicate that
the immunomodulatory activity of GP was not due to LPS
contamination.
4. Discussion
Extracts from glycyrrhiza have been used as a traditional
remedy and possess various pharmacological functions. For
Figure 6 Effects of GP on IL-6 secretion from mice macro-
phages in vitro. (A) Peritoneal macrophages were treated with
GP or LPS (2 g/mL) for 72 h. (B) Macrophages were isolated from
BALB/c mice, and cultured in 96-well flat-bottomed plate with
or without GP (100 g/mL) for 072 h. Culture media were
analyzed for IL-6 by a commercial ELISA kit. Each value
represents the mean S.D., (n = 3). P 0.01 vs. control.
48
Figure 7 Effects of GP on IL-12 secretion from mice macro-
phages in vitro. (A) Peritoneal macrophages were treated with GP
or LPS (2 g/mL) for 72 h. (B) Macrophages were cultured in 96-
well flat-bottomed plate with GP (100 g/mL) for 072 h. Culture
media were analyzed for IL-12 by a commercial ELISA kit. Each
value represents the mean S.D., (n = 3). P 0.01 vs. control.
example, polysaccharides extracted from Glycyrrhiza ura-
lensis Fish enhance the expression of bax protein and
decreases that of bac-2, p53 protein of sarcoma 180 cells
[34], and exhibits anti-complementary and mitogenic activ-
ities [35]. In the present report, we demonstrated poly-
saccharides of glycyrrhiza have immuno-modulatory
properties that enhance macrophage functions and suggest
that this may contribute, at least in part, to the therapeutic
potential of glycyrrhiza.
Differences in activities are generally related to solubility
in water, molecular size, branching frequency and forms. It is
believed that structural features such as (1 3) linkage in
the main chain of the glucan and additional (1 6) branch
points are needed for anticancer action [6]. We have isolated
polysaccharides fraction from glycyrrhiza and provided main
structural and pharmacological characterization. By analyz-
ing of IR spectrum and observing the helical structure of
glycyrrhiza polysaccharide using atomic force microscope
[27], the structure of GP did not contain one main core, but
three main cores. The main chain of GP was composed of -
1,3-linked D-galactose residues and/or -1,4- linked D-
glucose. The bioactivities of GP depend on its intricate and
helical structure. Recent studies indicated that GP includes a
backbone chain composed of -1,3-linked D-galactose
residues, three-fifths of the galactose units in the backbone
carry side chains composed of beta-1,3- and beta-1,6-linked
D-galactosyl residues at position 6 [23], this structure is
important for the anti-complementary activity. Crude poly-
saccharide fraction obtained from the shoot and hairy root of
Glycyrrhizae sp. induced nitric oxide production by murine
A. Cheng et al.
peritoneal macrophages in vitro under aseptic conditions
[22]. Chemical analysis revealed that LPS-like molecules
were not present in all preparations. Similarly, we found
polysaccharides derived from glycyrrhiza uralensis Fish also
induced and increased nitric oxide production by peritoneal
macrophages from BALB/C mice. Yang et al. [21] reported
glycyrrhiza polysaccharides could activate peritoneal macro-
phages and increase IL-1 production. Likewise, we found
glycyrrhiza-derived polysaccharide induced IL-6, IL-12 pro-
duction when tested over a wide concentration range. It will
be interesting to determine whether these structural
features contribute to the immunomodulatory activity of GP.
Macrophages are the major source of IL-1, IL-6, IL-12 and
NO, and participate as major effector cells in resistance
against infectious agents and tumor cells. Macrophages are
activated to become cytotoxic by a set of cytokine signals.
Therefore, in the present study, we examined whether GP
activated macrophages to induce effector molecules such as
cytokines, NO and O2 . GP was shown to stimulate macro-
phages to produced IL-1, IL-6 and IL-12 in a dose- and time-
dependent manner. IL-1 has direct in vitro cytostatic and
cytocidal effects; IL-6 is also considered as a major immune
and inflammatory mediator; IL-12 produced by macrophages
enhances T-cell responsiveness [1]. The above three cyto-
kines are related to each other in that they are coordinately
released from activated macrophages, and that IL-1 can
induce IL-6. However, IL-6 does not induce IL-1, but rather
suppresses their production by macrophages [1]. Polysac-
charides directly triggered IL-6 protein production, but not
through stimulation of IL-1. IL-12 induces IFN- secretion and
promotes growth of activated T cell and NK cells [1]. In the
present paper, we only studied the effects of GP on the
protein of IL-1, IL-6, IL-12 and NO. Further studies are
needed to investigate the mRNA expressions of IL-1, IL-6, IL-
12 and NO, and define the relative roles of these cytokines in
conferring their biological activity.
NO is a gaseous molecule synthesized from L-arginine by
nitric oxide synthase (NOS). NO is known to play a key role
Figure 8 Effects of polymyxin B-treatment on NO production.
LPS (2 g/mL), GP (100 g/mL), or LPS (2 g/mL) and GP
(100 g/mL) mixture was added to polymyxin B-affinity column,
incubated overnight at 4 C, eluted from the column by
centrifugation, and then added to the cultures of macrophages
(final, 1:80 dilution) for 48 h. Filled bars represent NO inducing
activity without polymyxin B treatment.
Macrophage immunomodulatory activity of polysaccharides isolated from Glycyrrhiza uralensis fish
during the course of infections [36]. In the present study we
observed that GP stimulated macrophages to produce NO,
which has been shown to be the principal effector molecule
produced by macrophages for cytotoxic activity. NO can be
used as a quantitative index of macrophage activation [37].
These results are reminiscent of similar properties shown by
LPS that also stimulates NO production by macrophages. The
non-activated macrophages produce very low amount of NO.
The possible roles of macrophages in the anti-tumor ac-
tivity of GP, since NO is related to cytolytic function of
macrophages against a variety of tumors [38]. Since endo-
toxin is widely present in many plant materials, potential
contamination of endotoxin is always a concern for the high
molecular weight components isolated from plants. Endo-
toxin is also a strong activator of macrophages, which may
contributes to the GP effects we have observed. The
macrophage-activating activity of GP, however, was not
due to endotoxin contamination as shown by the polymyxin
B-treatment experiments (Fig. 8).
Polysaccharides have been examined for their ability to
trigger O2 production by macrophages. Macrophages from GP-
treated animals were shown to be activated in the absence of
PMA, as evidenced by the higher O2 production than that of
macrophages from untreated mice starting at 60 min in our
time course study. Similar results were also observed in the
presence of PMA, with higher O2 production observed at
30 min. In addition, the effects of GP on macrophages O2
production in the absence or presence of PMA were a dose- and
time-dependent. Since phagocytes act as regulatory and
effector cells in the immune system, an enhancement of the
phagocyte function suggests a potential application of GP
therapy in treating microbial infections and cancer.
In conclusion, increased production of cytokines by
macrophages likely contributes to the therapeutic effects
of GP, and our data have provided further evidence that
glycyrrhiza derived polysaccharides had potent immunomo-
dulatory properties. Future work will focus on the delinea-
tion of underlying mechanisms and signaling cascades
involved in target tissues.
Acknowledgements
The authors acknowledge Dr. Hu Chingyuan (Research
College of Tropical Agriculture and Human Resources,
University of Hawaii, USA) for his help in manuscript revision,
and thank Dean Zhang Xiumei (Institute the Animal Science
and Veterinary Medicine, Shandong Academy of Agriculture
Science, China) for her help.
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