# A Water-Soluble Polysaccharide from the Fruit Bodies ofBulgaria inquinans (Fries) and Its Anti-Malarial Activity

Authors: Hongtao Bi; Han Han; Zonghong Li; Weihua Ni; Yan Chen; Jingjing Zhu; Tingting Gao; Miao Hao; Yifa Zhou
Journal: Evidence-Based Complementary and Alternative Medicine
 (2011)

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## Abstract

A water-soluble polysaccharide (BIWS-4b) was purified from the fruit bodies ofBulgaria inquinans (Fries). It is composed of mannose (27.2%), glucose (15.5%) and galactose (57.3%). Its molecular weight was estimated to be 7.4 kDa (polydispersity index, Mw/Mn: 1.35). Structural analyses indicated that BIWS-4b mainly contains (1→6)-linked, (1→5)-linked and (1→5,6)-linked β-Galf units; (1→4)-linked and non-reducing terminal β-Glcp units; and (1→2)-linked, (1→6)-linked, (1→2,6)-linked and non-reducing terminal α-Manp units. When examined by the 4-day method and in a prophylactic assay in mice, BIWS-4b exhibited markedly suppressive activity against malaria while enhancing the activity of artesunate. Immunological tests indicated that BIWS-4b significantly enhanced macrophage phagocytosis and splenic lymphocyte proliferation in malaria-bearing mice and normal mice. The anti-malarial activity of BIWS-4b might be intermediated by enhancing immune competence and restoring artesunate-suppressed immune function. Thus, BIWS-4b is a potential adjuvant of anti-malaria drugs.


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## Body

## 1.
Introduction

Many fungi are used as traditional medicines in the
treatment of various human diseases such as hepatitis, hypertension,
hypercholesterolemia and gastric cancer [1–4]. Recently, fungal polysaccharides have
received considerable attention as an important class of bioactive substances
because of their potent biological and pharmacological activities, especially
immunological and antitumor activities [5–8]. Discovery and evaluation of new bioactive
polysaccharides from medicinal fungi has emerged as one of the hot research
fields in chemistry and biology. Due to their immunological activities, some
fungal polysaccharides could protect hosts from microbes and parasites such as
malaria [9].The non-lichenized
ascomyceteBulgaria inquinans (Fries) is an edible
wood-inhabiting ascomycete that grows on freshly felled oak and commonly found
in the Changbai Mountain area of China. It has been used as food and folk
antitumor medicine for a long time. Several small molecule compounds isolated
from the fruit bodies of B. inquinans (Fries) have been found
to have antitumor [10], antipruritic and
antierythema activities [11]. However, the
polysaccharides from B. inquinans (Fries) have not been
thoroughly studied. In a previous study, we fractionated polysaccharides from
B. inquinans (Fries) and a low molecular weight
β-(1→6)-d-glucan was
characterized [12]. In the present article, we
describe the characterization of the more complex heteropolysaccharide fraction
BIWS-4b from B. inquinans (Fries) and its anti-malarial
activity in vivo as well as a possible mechanism of
action.


## 2. Methods

### 2.1. Materials and
Chemicals

Fruit bodies ofB. inquinans (Fries) were
collected from Changbai Mountain, China and identified by Professor Hongxing
Xiao at Northeast Normal University in Changchun, China. A voucher specimen
(No. 20070802) was deposited at the School of Life Sciences, Northeast Normal
University.Artesunate was purchased from Guilin Pharmaceutical Co.,
Ltd., China. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
(MTT), concanavalin A (ConA), lipopolysaccharide (LPS), Sepharose CL-6B and
Sephadex G-75 were purchased from Sigma Chemical Co. RPMI-1640 medium was
obtained from Gibco Invitrogen Co. The complete RMPI-1640 medium used for
immunological testing had a pH of 7.4 and was supplemented with penicillin
(100 IU mL−1), streptomycin (100 μg ml−1) and 10%
fetal bovine serum (FBS). All other reagents were of analytical grade and made
in China.


### 2.2.
Animals

An equal number of male and female ICR mice (Grade II, 6–8
weeks old), weighing 18–22 g, were purchased from the Pharmacology Experimental
Center of Jilin University (approval number: SCXK (Jilin) 2002-0002, Changchun,
China) and acclimatized for one week prior to use. All mice were housed under
standard conditions at 24± 1°C with
humidity of 50 ± 10% and a 12/12 h
light/dark cycle. Rodent laboratory chow pellets and tap water were supplied
ad libitum. All procedures were in strict accordance with PR
China legislation and with the guidelines established by the School of Life
Sciences of Northeast Normal University regarding the use and care of
laboratory animals. The protocol was approved by the University’s Committee for
Animal Experiments and Science & Technology Department of Jilin Province
(approval number: SCXK (Jilin) 2003-0001).


### 2.3. Isolation and Purification
of BIWS-4b

The isolation of polysaccharides from fruit bodies ofB. inquinans (Fries) was carried out as previously described
by our group [12]. In brief, dried fruit bodies
of B. inquinans (Fries) were extracted with 95% ethanol under
reflux for 12 h to remove hydrophobic compounds and then extracted three times
with hot distilled water (6 h for each extraction, 90–95°C,
1 : 20 w/v). The extracts were combined and concentrated to one-tenth of the
original volume, and then ethanol was added to the concentrated extracts up to
80% to precipitate the crude polysaccharides. Crude polysaccharides were
collected by centrifugation, and then treated with Sevag reagent [13] to remove free proteins to give the polysaccharide
fraction (BIW). The aqueous solution of BIW (10% w/v) was frozen at
–20°C and then allowed to thaw slowly at 4°C, which
yielded the soluble fraction (BIWS) and insoluble fraction (BIWP). BIWP was
fractionated and characterized in our previous study [12]. In the present study, BIWS was dissolved in water
(10% w/v), and then fractionated by gradient precipitation in 30, 50, 70 and
90% ethanol resulting in four fractions BIWS-1, BIWS-2, BIWS-3 and BIWS-4,
respectively. BIWS-4 was further fractionated on a column (100 × 1.5 cm) of Sephadex G-75
and eluted with 0.15 M NaCl to give two fractions: BIWS-4a collected in the
void volume and BIWS-4b collected as a main portion. After dialysis in tubing
(Mw cut-off 3.5 × 103 Da for
global protein), the fractions were concentrated and lyophilized to give the
pure polysaccharide fraction BIWS-4b. The extraction and purification procedure
was shown in Figure 1. All gel filtration
chromatographies were monitored by assaying carbohydrate content.Figure 1
Extraction and purification procedure of BIWS-4b.


### 2.4. Analytical Methods

The total carbohydrate content was determined by the
phenol-H2SO4 method using glucose as a standard [14]. Protein content was determined in a Bradford
assay using bovine serum albumin as the standard [15]. Contaminant endotoxin was analyzed in a limulus amebocyte lysate
(LAL) assay using an E-TOXATE kit (Sigma, St. Louis, USA) according to the
manufacturer’s instructions.Monosaccharide analysis was performed as
described by Honda et al. [16]. Briefly, sample
(2 mg) was hydrolyzed with 2 M CF3COOH (1.0 ml) at 120°C
for 3 h. The hydrolyzed-products (monosaccharides) were derivatized with 0.5 M
1-phenyl-3-methyl-5- pyrazolone (PMP) and 0.3 M NaOH. After neutralization with
0.3 M HCl, the PMP-derivatives were analyzed in a Shimadzu 10Avp HPLC system
equipped with a Shim-pak VP-ODS column (150  ×  4.6 mm i.d.) with guard
column and monitored by UV absorbance at 245 nm.The specific rotation
was measured at 20± 1°C with an
automatic polarimeter (Model WZZ-2B, China). UV-Vis absorbance spectra were
recorded with a UV-Vis spectrophotometer (Model SP-754, China). FT-IR spectra
were obtained on a Nicolet 560 FT-IR spectrometer with DTGS detector in a range
of 400–4000 cm−1. The samples were ground with KBr powder and then
pressed into 1 mm pellets for FT-IR measurements.The13C NMR
spectrum was recorded using a Bruker 5 mm Broadband Observe probe at
20°C in a Bruker Avance 600 MHz spectrometer (Germany) operating at
150 MHz. Sample (20 mg) was dissolved in D2O (99.8%, 0.5 ml),
freeze-dried, redissolved in D2O (0.5 ml) and centrifuged to remove
excess sample. The experiment was recorded using standard Bruker software.


### 2.5. Homogeneity and
Molecular Weight Determination

Homogeneity and molecular weight were
determined in a Shimadzu 10Avp HPLC system equipped with a 10Avp Pump and
RID-10A Refractive Index Detector. The system was linked to the gel filtration
column TSK-G3000 PWXL and sample was eluted with 0.2 M NaCl at a
flow rate of 0.55 ml min−1 at 35.0 ± 0.1°C. The
column was calibrated by standard dextrans (50, 25, 12, 5 and 1 kDa) using
linear regression. Sample concentration upon injection was 5 mg ml−1
and approximately 20 μl was injected.


### 2.6. Methylation
Analysis

The methylation analysis was carried out according to the
method of Needs and Selvendran [17]. In brief,
BIWS-4b (10 mg) was dissolved in DMSO (1.5 ml) and then methylated by treatment
with a NaOH/DMSO suspension (1 ml) and iodomethane (1 ml). The methylated
polysaccharide was extracted with CHCl3 and then the solvent was
removed by vacuum evaporation. The progress of methylation was followed by the
disappearance of the –OH band (3200–3400 cm−1) in the FT-IR
spectrum. The per-O-methylated polysaccharides were hydrolyzed
subsequently by HCOOH (85%, 0.5 ml) for 4 h and CF3COOH (2 M, 1 ml)
for 6 h at 100°C. Partially methylated sugars in the hydrolysate
were reduced by NaBH4 and then acetylated with pyridine (0.5 ml) and
acetic anhydride (0.5 ml) at 90°C for 1 h. The resulting mixture of
alditol acetates was analyzed by GC-MS. GC-MS was carried out on an Agilent
6890N-5975 instrument equipped with a HP-5 column.


### 2.7. Preliminary Anti-Malaria
Testing In Vivo

#### 2.7.1.
Parasite Inoculation

ThePlasmodium yoelii strain
17XL was a gift from Prof. Cao Yaming (Department of Immunology, China Medical
University) and blood stage parasites were stored in liquid nitrogen. The
standard inoculum consisted of 5 ×107 ml−1 P. yoelii
parasitized erythrocytes from a donor mouse, which was used to infect mice
intraperitoneally.


#### 2.7.2. Suppressive Activity on Early Infection (4-Day Test)

Suppression of early infection was evaluated using a 4-day test described by
Peters and Robinson [18]. Briefly, after
inoculating with 0.2 ml of standard inoculum, the mice were randomly divided
into six groups of six mice each. The polysaccharide-treated groups were given
BIWS-4b of different dosages (50, 100 and 200 mg kg−1), and the
co-treated group was given the mixture of BIWS-4b (100 mg kg−1) and
artesunate (14 mg kg−1). A negative control group was administered
physiological saline and positive control groups were treated with artesunate
at doses of 14 or 28 mg kg−1. The drugs were orally administered to
mice in 0.2 ml doses daily for 4 consecutive days (Days 0–3). On
Day 4, blood smears were generated from tail blood and stained with Giemsa
stain. Drug activity was assessed by evaluating the smears under a microscope.
Parasitemia (%) was calculated by dividing the number of parasitized
erythrocytes by the total number of erythrocytes. Average chemosuppression (%)
was calculated as 100  ×  [(A – B)/A],
where A is the average parasitemia of the negative control
group and B is the average parasitemia of the test group.


#### 2.7.3. Prophylactic
Activity on Residual Infection (Prophylactic Test)

Prophylactic
activity was assessed according to the method described by Peters [19]. Mice were randomly divided into groups of six
mice each and orally received BIWS-4b or physiological saline (negative
control) for 4, 6, 8 or 10 consecutive days prior to infection. Twenty-four
hours after the final administration of drug or saline, the mice were
inoculated with P. yoelii. Seventy-two hours after
inoculation, parasitemia levels were assessed in blood smears from tail blood
of each mouse, and parasitemia (%) and average chemosuppression (%) were
calculated.


### 2.8. Evaluation of Immunostimulating Activity In Vivo

#### 2.8.1. Drug
Administration

The mice were randomly divided into groups of six
mice each: a control group (administered physiological saline) and 100 and
200 mg kg−1 BIWS-4b groups. The drugs were given orally in doses of
0.2 ml for 4, 6, 8 or 10 consecutive days. Twenty-four hours after the final
administration of drug or saline, mice in the malaria-bearing groups were
inoculated with P. yoelii while the control group was used to
assess the macrophage phagocytosis and lymphocyte proliferation activities of
BIWS-4b in normal mice. Seventy-two hours after inoculation, the macrophage
phagocytosis and lymphocyte proliferation activities of BIWS-4b in
malaria-bearing mice were evaluated.


#### 2.8.2. Phagocytosis of
Macrophage Assay

Chicken red blood cells (CRBC) were used to assess
macrophage phagocytosis as described by Yang et al. [20]. Briefly, on the last day, 1 ml of 20% (v/v) CRBC was
intraperitoneally injected into each mouse, and the mice were sacrificed 30 min
later. Physiological saline (2 ml) was injected into the abdominal cavity, and
then 1 ml fluid was collected to make a smear from each mouse. After incubating
at 37°C for 30 min in a humidified 5% CO2 incubator, the
smears were centrifuged to remove the supernatant, and the macrophages were
fixed with methanol and stained by Giemsa-Wright for 7–10 min. The phagocytosis
index was measured by counting the number of phagocytosed CRBC per every 100
macrophage cells, and the phagocytosis rate was measured by counting the number
of macrophages containing CRBC per every 100 macrophage cells.


#### 2.8.3. Lymphocyte
Proliferation Assay

Spleens were aseptically extirpated from the
immunized mice. Spleen single cell suspensions were pooled in ice-cold Hank’s
solution by filtering through sieve mesh. Spleen cells were depleted of
erythrocytes in Tris2NH4Cl (0.16 M,
Tris2NH4Cl, pH 7.2), due to low-osmosis, followed by two
washes in Hank’s solution and resuspending in complete RPMI 1640 medium.
Splenocyte activity was measured above 95% as assessed by the trypan blue dye
exclusion method.The lymphocyte proliferation assay was followed as
described by Lee et al. [21] with slight
modification. Briefly, spleen lymphocytes were seeded into 96-well flat-bottom
microtiter plates at 5 ×106 cells ml−1 and cultured in RPMI 1640
medium contained ConA (5.0 μg ml−1) or LPS
(10.0 μg ml−1). The
plates were incubated at 37°C in a humidified atmosphere with 5%
CO2. After 44 h, 20 μl of MTT solution
(5 μg ml−1) was
added to each well and the cells were incubated for an additional 4 h. After
aspirating the supernatant from the wells, 100 μl of DMSO was added to
dissolve formazan crystal. The absorbance at 570 nm was measured using a
Bio-Rad microplate reader (Model 550, USA).


### 2.9. Statistical
Analysis

All data were expressed as the mean± SD of six replicates and
examined for statistical significance with Student’s t-tests.
A result is considered statistically significant when P<.05. One-way ANOVA
was conducted to assess significant differences among the treatments as a whole
in order to avoid the error inherent in performing multiple
t-tests.


## 2.1. Materials and
Chemicals

Fruit bodies ofB. inquinans (Fries) were
collected from Changbai Mountain, China and identified by Professor Hongxing
Xiao at Northeast Normal University in Changchun, China. A voucher specimen
(No. 20070802) was deposited at the School of Life Sciences, Northeast Normal
University.Artesunate was purchased from Guilin Pharmaceutical Co.,
Ltd., China. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
(MTT), concanavalin A (ConA), lipopolysaccharide (LPS), Sepharose CL-6B and
Sephadex G-75 were purchased from Sigma Chemical Co. RPMI-1640 medium was
obtained from Gibco Invitrogen Co. The complete RMPI-1640 medium used for
immunological testing had a pH of 7.4 and was supplemented with penicillin
(100 IU mL−1), streptomycin (100 μg ml−1) and 10%
fetal bovine serum (FBS). All other reagents were of analytical grade and made
in China.


## 2.2.
Animals

An equal number of male and female ICR mice (Grade II, 6–8
weeks old), weighing 18–22 g, were purchased from the Pharmacology Experimental
Center of Jilin University (approval number: SCXK (Jilin) 2002-0002, Changchun,
China) and acclimatized for one week prior to use. All mice were housed under
standard conditions at 24± 1°C with
humidity of 50 ± 10% and a 12/12 h
light/dark cycle. Rodent laboratory chow pellets and tap water were supplied
ad libitum. All procedures were in strict accordance with PR
China legislation and with the guidelines established by the School of Life
Sciences of Northeast Normal University regarding the use and care of
laboratory animals. The protocol was approved by the University’s Committee for
Animal Experiments and Science & Technology Department of Jilin Province
(approval number: SCXK (Jilin) 2003-0001).


## 2.3. Isolation and Purification
of BIWS-4b

The isolation of polysaccharides from fruit bodies ofB. inquinans (Fries) was carried out as previously described
by our group [12]. In brief, dried fruit bodies
of B. inquinans (Fries) were extracted with 95% ethanol under
reflux for 12 h to remove hydrophobic compounds and then extracted three times
with hot distilled water (6 h for each extraction, 90–95°C,
1 : 20 w/v). The extracts were combined and concentrated to one-tenth of the
original volume, and then ethanol was added to the concentrated extracts up to
80% to precipitate the crude polysaccharides. Crude polysaccharides were
collected by centrifugation, and then treated with Sevag reagent [13] to remove free proteins to give the polysaccharide
fraction (BIW). The aqueous solution of BIW (10% w/v) was frozen at
–20°C and then allowed to thaw slowly at 4°C, which
yielded the soluble fraction (BIWS) and insoluble fraction (BIWP). BIWP was
fractionated and characterized in our previous study [12]. In the present study, BIWS was dissolved in water
(10% w/v), and then fractionated by gradient precipitation in 30, 50, 70 and
90% ethanol resulting in four fractions BIWS-1, BIWS-2, BIWS-3 and BIWS-4,
respectively. BIWS-4 was further fractionated on a column (100 × 1.5 cm) of Sephadex G-75
and eluted with 0.15 M NaCl to give two fractions: BIWS-4a collected in the
void volume and BIWS-4b collected as a main portion. After dialysis in tubing
(Mw cut-off 3.5 × 103 Da for
global protein), the fractions were concentrated and lyophilized to give the
pure polysaccharide fraction BIWS-4b. The extraction and purification procedure
was shown in Figure 1. All gel filtration
chromatographies were monitored by assaying carbohydrate content.Figure 1
Extraction and purification procedure of BIWS-4b.


## 2.4. Analytical Methods

The total carbohydrate content was determined by the
phenol-H2SO4 method using glucose as a standard [14]. Protein content was determined in a Bradford
assay using bovine serum albumin as the standard [15]. Contaminant endotoxin was analyzed in a limulus amebocyte lysate
(LAL) assay using an E-TOXATE kit (Sigma, St. Louis, USA) according to the
manufacturer’s instructions.Monosaccharide analysis was performed as
described by Honda et al. [16]. Briefly, sample
(2 mg) was hydrolyzed with 2 M CF3COOH (1.0 ml) at 120°C
for 3 h. The hydrolyzed-products (monosaccharides) were derivatized with 0.5 M
1-phenyl-3-methyl-5- pyrazolone (PMP) and 0.3 M NaOH. After neutralization with
0.3 M HCl, the PMP-derivatives were analyzed in a Shimadzu 10Avp HPLC system
equipped with a Shim-pak VP-ODS column (150  ×  4.6 mm i.d.) with guard
column and monitored by UV absorbance at 245 nm.The specific rotation
was measured at 20± 1°C with an
automatic polarimeter (Model WZZ-2B, China). UV-Vis absorbance spectra were
recorded with a UV-Vis spectrophotometer (Model SP-754, China). FT-IR spectra
were obtained on a Nicolet 560 FT-IR spectrometer with DTGS detector in a range
of 400–4000 cm−1. The samples were ground with KBr powder and then
pressed into 1 mm pellets for FT-IR measurements.The13C NMR
spectrum was recorded using a Bruker 5 mm Broadband Observe probe at
20°C in a Bruker Avance 600 MHz spectrometer (Germany) operating at
150 MHz. Sample (20 mg) was dissolved in D2O (99.8%, 0.5 ml),
freeze-dried, redissolved in D2O (0.5 ml) and centrifuged to remove
excess sample. The experiment was recorded using standard Bruker software.


## 2.5. Homogeneity and
Molecular Weight Determination

Homogeneity and molecular weight were
determined in a Shimadzu 10Avp HPLC system equipped with a 10Avp Pump and
RID-10A Refractive Index Detector. The system was linked to the gel filtration
column TSK-G3000 PWXL and sample was eluted with 0.2 M NaCl at a
flow rate of 0.55 ml min−1 at 35.0 ± 0.1°C. The
column was calibrated by standard dextrans (50, 25, 12, 5 and 1 kDa) using
linear regression. Sample concentration upon injection was 5 mg ml−1
and approximately 20 μl was injected.


## 2.6. Methylation
Analysis

The methylation analysis was carried out according to the
method of Needs and Selvendran [17]. In brief,
BIWS-4b (10 mg) was dissolved in DMSO (1.5 ml) and then methylated by treatment
with a NaOH/DMSO suspension (1 ml) and iodomethane (1 ml). The methylated
polysaccharide was extracted with CHCl3 and then the solvent was
removed by vacuum evaporation. The progress of methylation was followed by the
disappearance of the –OH band (3200–3400 cm−1) in the FT-IR
spectrum. The per-O-methylated polysaccharides were hydrolyzed
subsequently by HCOOH (85%, 0.5 ml) for 4 h and CF3COOH (2 M, 1 ml)
for 6 h at 100°C. Partially methylated sugars in the hydrolysate
were reduced by NaBH4 and then acetylated with pyridine (0.5 ml) and
acetic anhydride (0.5 ml) at 90°C for 1 h. The resulting mixture of
alditol acetates was analyzed by GC-MS. GC-MS was carried out on an Agilent
6890N-5975 instrument equipped with a HP-5 column.


## 2.7. Preliminary Anti-Malaria
Testing In Vivo

### 2.7.1.
Parasite Inoculation

ThePlasmodium yoelii strain
17XL was a gift from Prof. Cao Yaming (Department of Immunology, China Medical
University) and blood stage parasites were stored in liquid nitrogen. The
standard inoculum consisted of 5 ×107 ml−1 P. yoelii
parasitized erythrocytes from a donor mouse, which was used to infect mice
intraperitoneally.


### 2.7.2. Suppressive Activity on Early Infection (4-Day Test)

Suppression of early infection was evaluated using a 4-day test described by
Peters and Robinson [18]. Briefly, after
inoculating with 0.2 ml of standard inoculum, the mice were randomly divided
into six groups of six mice each. The polysaccharide-treated groups were given
BIWS-4b of different dosages (50, 100 and 200 mg kg−1), and the
co-treated group was given the mixture of BIWS-4b (100 mg kg−1) and
artesunate (14 mg kg−1). A negative control group was administered
physiological saline and positive control groups were treated with artesunate
at doses of 14 or 28 mg kg−1. The drugs were orally administered to
mice in 0.2 ml doses daily for 4 consecutive days (Days 0–3). On
Day 4, blood smears were generated from tail blood and stained with Giemsa
stain. Drug activity was assessed by evaluating the smears under a microscope.
Parasitemia (%) was calculated by dividing the number of parasitized
erythrocytes by the total number of erythrocytes. Average chemosuppression (%)
was calculated as 100  ×  [(A – B)/A],
where A is the average parasitemia of the negative control
group and B is the average parasitemia of the test group.


### 2.7.3. Prophylactic
Activity on Residual Infection (Prophylactic Test)

Prophylactic
activity was assessed according to the method described by Peters [19]. Mice were randomly divided into groups of six
mice each and orally received BIWS-4b or physiological saline (negative
control) for 4, 6, 8 or 10 consecutive days prior to infection. Twenty-four
hours after the final administration of drug or saline, the mice were
inoculated with P. yoelii. Seventy-two hours after
inoculation, parasitemia levels were assessed in blood smears from tail blood
of each mouse, and parasitemia (%) and average chemosuppression (%) were
calculated.


## 2.7.1.
Parasite Inoculation

ThePlasmodium yoelii strain
17XL was a gift from Prof. Cao Yaming (Department of Immunology, China Medical
University) and blood stage parasites were stored in liquid nitrogen. The
standard inoculum consisted of 5 ×107 ml−1 P. yoelii
parasitized erythrocytes from a donor mouse, which was used to infect mice
intraperitoneally.


## 2.7.2. Suppressive Activity on Early Infection (4-Day Test)

Suppression of early infection was evaluated using a 4-day test described by
Peters and Robinson [18]. Briefly, after
inoculating with 0.2 ml of standard inoculum, the mice were randomly divided
into six groups of six mice each. The polysaccharide-treated groups were given
BIWS-4b of different dosages (50, 100 and 200 mg kg−1), and the
co-treated group was given the mixture of BIWS-4b (100 mg kg−1) and
artesunate (14 mg kg−1). A negative control group was administered
physiological saline and positive control groups were treated with artesunate
at doses of 14 or 28 mg kg−1. The drugs were orally administered to
mice in 0.2 ml doses daily for 4 consecutive days (Days 0–3). On
Day 4, blood smears were generated from tail blood and stained with Giemsa
stain. Drug activity was assessed by evaluating the smears under a microscope.
Parasitemia (%) was calculated by dividing the number of parasitized
erythrocytes by the total number of erythrocytes. Average chemosuppression (%)
was calculated as 100  ×  [(A – B)/A],
where A is the average parasitemia of the negative control
group and B is the average parasitemia of the test group.


## 2.7.3. Prophylactic
Activity on Residual Infection (Prophylactic Test)

Prophylactic
activity was assessed according to the method described by Peters [19]. Mice were randomly divided into groups of six
mice each and orally received BIWS-4b or physiological saline (negative
control) for 4, 6, 8 or 10 consecutive days prior to infection. Twenty-four
hours after the final administration of drug or saline, the mice were
inoculated with P. yoelii. Seventy-two hours after
inoculation, parasitemia levels were assessed in blood smears from tail blood
of each mouse, and parasitemia (%) and average chemosuppression (%) were
calculated.


## 2.8. Evaluation of Immunostimulating Activity In Vivo

### 2.8.1. Drug
Administration

The mice were randomly divided into groups of six
mice each: a control group (administered physiological saline) and 100 and
200 mg kg−1 BIWS-4b groups. The drugs were given orally in doses of
0.2 ml for 4, 6, 8 or 10 consecutive days. Twenty-four hours after the final
administration of drug or saline, mice in the malaria-bearing groups were
inoculated with P. yoelii while the control group was used to
assess the macrophage phagocytosis and lymphocyte proliferation activities of
BIWS-4b in normal mice. Seventy-two hours after inoculation, the macrophage
phagocytosis and lymphocyte proliferation activities of BIWS-4b in
malaria-bearing mice were evaluated.


### 2.8.2. Phagocytosis of
Macrophage Assay

Chicken red blood cells (CRBC) were used to assess
macrophage phagocytosis as described by Yang et al. [20]. Briefly, on the last day, 1 ml of 20% (v/v) CRBC was
intraperitoneally injected into each mouse, and the mice were sacrificed 30 min
later. Physiological saline (2 ml) was injected into the abdominal cavity, and
then 1 ml fluid was collected to make a smear from each mouse. After incubating
at 37°C for 30 min in a humidified 5% CO2 incubator, the
smears were centrifuged to remove the supernatant, and the macrophages were
fixed with methanol and stained by Giemsa-Wright for 7–10 min. The phagocytosis
index was measured by counting the number of phagocytosed CRBC per every 100
macrophage cells, and the phagocytosis rate was measured by counting the number
of macrophages containing CRBC per every 100 macrophage cells.


### 2.8.3. Lymphocyte
Proliferation Assay

Spleens were aseptically extirpated from the
immunized mice. Spleen single cell suspensions were pooled in ice-cold Hank’s
solution by filtering through sieve mesh. Spleen cells were depleted of
erythrocytes in Tris2NH4Cl (0.16 M,
Tris2NH4Cl, pH 7.2), due to low-osmosis, followed by two
washes in Hank’s solution and resuspending in complete RPMI 1640 medium.
Splenocyte activity was measured above 95% as assessed by the trypan blue dye
exclusion method.The lymphocyte proliferation assay was followed as
described by Lee et al. [21] with slight
modification. Briefly, spleen lymphocytes were seeded into 96-well flat-bottom
microtiter plates at 5 ×106 cells ml−1 and cultured in RPMI 1640
medium contained ConA (5.0 μg ml−1) or LPS
(10.0 μg ml−1). The
plates were incubated at 37°C in a humidified atmosphere with 5%
CO2. After 44 h, 20 μl of MTT solution
(5 μg ml−1) was
added to each well and the cells were incubated for an additional 4 h. After
aspirating the supernatant from the wells, 100 μl of DMSO was added to
dissolve formazan crystal. The absorbance at 570 nm was measured using a
Bio-Rad microplate reader (Model 550, USA).


## 2.8.1. Drug
Administration

The mice were randomly divided into groups of six
mice each: a control group (administered physiological saline) and 100 and
200 mg kg−1 BIWS-4b groups. The drugs were given orally in doses of
0.2 ml for 4, 6, 8 or 10 consecutive days. Twenty-four hours after the final
administration of drug or saline, mice in the malaria-bearing groups were
inoculated with P. yoelii while the control group was used to
assess the macrophage phagocytosis and lymphocyte proliferation activities of
BIWS-4b in normal mice. Seventy-two hours after inoculation, the macrophage
phagocytosis and lymphocyte proliferation activities of BIWS-4b in
malaria-bearing mice were evaluated.


## 2.8.2. Phagocytosis of
Macrophage Assay

Chicken red blood cells (CRBC) were used to assess
macrophage phagocytosis as described by Yang et al. [20]. Briefly, on the last day, 1 ml of 20% (v/v) CRBC was
intraperitoneally injected into each mouse, and the mice were sacrificed 30 min
later. Physiological saline (2 ml) was injected into the abdominal cavity, and
then 1 ml fluid was collected to make a smear from each mouse. After incubating
at 37°C for 30 min in a humidified 5% CO2 incubator, the
smears were centrifuged to remove the supernatant, and the macrophages were
fixed with methanol and stained by Giemsa-Wright for 7–10 min. The phagocytosis
index was measured by counting the number of phagocytosed CRBC per every 100
macrophage cells, and the phagocytosis rate was measured by counting the number
of macrophages containing CRBC per every 100 macrophage cells.


## 2.8.3. Lymphocyte
Proliferation Assay

Spleens were aseptically extirpated from the
immunized mice. Spleen single cell suspensions were pooled in ice-cold Hank’s
solution by filtering through sieve mesh. Spleen cells were depleted of
erythrocytes in Tris2NH4Cl (0.16 M,
Tris2NH4Cl, pH 7.2), due to low-osmosis, followed by two
washes in Hank’s solution and resuspending in complete RPMI 1640 medium.
Splenocyte activity was measured above 95% as assessed by the trypan blue dye
exclusion method.The lymphocyte proliferation assay was followed as
described by Lee et al. [21] with slight
modification. Briefly, spleen lymphocytes were seeded into 96-well flat-bottom
microtiter plates at 5 ×106 cells ml−1 and cultured in RPMI 1640
medium contained ConA (5.0 μg ml−1) or LPS
(10.0 μg ml−1). The
plates were incubated at 37°C in a humidified atmosphere with 5%
CO2. After 44 h, 20 μl of MTT solution
(5 μg ml−1) was
added to each well and the cells were incubated for an additional 4 h. After
aspirating the supernatant from the wells, 100 μl of DMSO was added to
dissolve formazan crystal. The absorbance at 570 nm was measured using a
Bio-Rad microplate reader (Model 550, USA).


## 2.9. Statistical
Analysis

All data were expressed as the mean± SD of six replicates and
examined for statistical significance with Student’s t-tests.
A result is considered statistically significant when P<.05. One-way ANOVA
was conducted to assess significant differences among the treatments as a whole
in order to avoid the error inherent in performing multiple
t-tests.


## 3. Results

### 3.1.
Isolation and Purification of BIWS-4b

Fruit bodies ofB.
inquinans (Fries) were treated with hot water, and extracted
polysaccharides were precipitated in ethanol. Deproteination by the Sevag
method gave an 8% yield of the polysaccharide (BIW) from dried fruit bodies.
BIW was composed of glucose, mannose and galactose in a molar ratio of
1.0 : 1.3  : 1.0, respectively. A freeze-thaw cycle of the polysaccharide
resulted in two fractions: BIWP (precipitate, 19%) and BIWS (supernatant, 63%).
Analysis of BIWP revealed glucan, which was further purified by Sephadex G-75
gel filtration chromatography to give a homogeneous
β-(1→6)-d-glucan
[12].BIWS consists of glucose, mannose
and galactose in a molar ratio of 1 :  1 : 1 and exhibits a wide molecular weight
distribution from 15 to 50 kDa. It was fractionated by gradient precipitation
in 30, 50, 70 and 90% ethanol, which resulted in the fractions BIWS-1, BIWS-2,
BIWS-3 and BIWS-4, respectively. BIWS-1, BIWS-2 and BIWS-3 showed wide
distributions on Sepharose CL-6B or Sephadex G-75 elution profiles, while
BIWS-4 gave two relatively narrow peaks on Sephadex G-75. Hence, BIWS-4 was
fractionated on a Sephadex G-75 column (1.5×  100 cm) into two
populations: BIWS-4a collected in the void elution volume (11% yield) and
BIWS-4b (72% yield) collected in the effective fractionation range as a main
portion. BIWS-4a is composed of glucose, mannose and galactose in a molar ratio
of 5.7 : 1.0 : 8.0. Because of its low yield and wide molecular weight
distribution, BIWS-4a was not studied further.


### 3.2. Analyses of BIWS-4b

BIWS-4b is a light brown powder and its total carbohydrate content was
determined to be 96% by the phenol-H2SO4 method. The UV
spectrum of BIWS-4b contained no peaks at 260 or 280 nm, and the Bradford assay
was negative, which indicated that BIWS-4b did not contain protein and nucleic
acid. A limulus amebocyte lysate (LAL) assay revealed less than 0.015 EU
(endotoxin units) mg−1, which indicates a negative result. Analysis
of sugar content showed that BIWS-4b consists of mannose (27.2%), glucose
(15.5%) and galactose (57.3%). BIWS-4b gives a narrow symmetric peak by HPSEC
(Figure 2), and its molecular weight was
estimated to be 7.4 kDa (polydispersity index, Mw/Mn: 1.35), which suggests
that BIWS-4b is a homogenous polysaccharide fraction.Figure 2
HPSEC elution profile of
BIWS-4b.The specific rotation measurement of BIWS-4b is [α]D20 –51.9° (c 0.2, H2O). The
high negative rotation suggests the dominating presence of
β-glycosidic linkages. The
FT-IR spectrum of BIWS-4b (Figure 3) shows absorption
of
β-type glycosidic linkages
at 875.5 cm−1. The other absorption bands at 3392.2 cm−1
(hydroxyl stretching vibration), 2933.3 cm−1 (C–H stretching
vibration), and 1647.0 cm−1 (bound water) were from the
corresponding sugar residues. Methylation products were sequentially acetylated
and reduced to give seven methylated alditol acetates (Figure 4). This procedure revealed that galactosyl residues
were (1→6)-linked, (1→5)-linked and (1→5,6)-linked
Galf units; glucosyl residues were (1→4)-linked and non-reducing
terminal Glcp units; mannosyl residues were (1→2)-linked, (1→6)-linked, (1→2,6)-linked and
non-reducing terminal Manp units. The ratios of each residue
are listed in Table 1.Table 1
Methylation analysis of BIWS-4b.
Partially methylated glycitolMolar ratioaRetention time (min)Linkage type1,4,5-Tri-acetyl-2,3,6-tri-O-methyl galactitol4.617.95→5)-Galf-(1→1,4,5,6-Tetra-acetyl-2,3-di-O-methyl galactitol3.117.88→5,6)-Galf-(1→1,4,6-Tri-acetyl-2,3,5-tri-O-methyl galactitol7.618.94→6)-Galf-(1→1,4,5-Tri-acetyl-2,3,6-tri-O-methyl glucitol2.218.32→4)-Glcp-(1→1,5-Di-acetyl-2,3,4,6-tetra-O-methyl glucitol2.617.11Glcp-(1→1,2,5-Tri-acetyl-3,4,6-tri-O-methyl mannitol2.417.84→2)-Manp-(1→1,5-Tri-acetyl-2,3,4,6-tri-O-methyl mannitol1.017.06Manp-(1→1,2,5,6-Tetra-acetyl-3,4-di-O-methyl mannitol1.218.82→2,6)-Manp-(1→1,5,6-Tri-acetyl-2,3,4-tri-O-methyl mannitol2.918.72→6)-Manp-(1→aRelative molar ratio, calculated from the ratio of peak area.Figure 3
FT-IR spectra: (a) BIWS-4b and (b) partially
methylated BIWS-4b.Figure 4
GC profile of BIWS-4b in
methylation analysis.The
structural features of BIWS-4b were further analyzed by13C NMR;
assignments of carbon atom signals are listed in
Table 2 and Figure 5.
According to NMR data in the literatures [22–28], the
anomeric carbon signals at
δ 107.73, 107.03 and 106.92
can be attributed to C-1 of three different linkages of
β-Galf
residues;
δ 102.43 and 102.19
correspond to C-1 of
β-Glcp and
δ~101.19 to 97.24
correspond to C-1 of
α-Manp.
These results were consistent with those of methylation analysis. Combining the
results from methylation and NMR analyses, we deduced that BIWS-4b was mainly
composed of (1→6)-linked, (1→5)-linked, and (1→5,6)-linked
β-Galf
units, as well as (1→4)-linked, non-reducing
terminal
β-Glcp
units, and (1→2)-linked, (1→6)-linked, (1→2,6)-linked and
non-reducing terminal
α-Manp
units.Table 2
13C NMR chemical shifts of BIWS-4b in D2O.
Residueδ (ppm)C-1C-2C-3C-4C-5C-6→5)-β-Galf-(1→107.7380.9376.8181.1875.4762.73→5,6)-β-Galf-(1→107.0381.4576.4082.5074.2068.01→6)-β-Galf-(1→106.9282.0076.6583.9869.8969.70→4)-β-Glcp-(1→102.4373.1874.3079.0974.8360.64β-Glcp-(1→102.1972.9674.2069.1275.8360.32α-Manp-(1→101.1969.1269.5466.5672.4060.10→2)-α-Manp-(1→99.4577.2568.7666.6772.4060.10→6)-α-Manp-(1→98.3769.1269.5466.5871.9564.89→2,6)-α-Manp-(1→97.2477.6269.4066.6771.9564.92Figure 5
13C NMR spectrum of BIWS-4b. Solvent:
D2O, temperature: 20°C.


### 3.3. Anti-Malarial Activity of
BIWS-4b

#### 3.3.1.
Anti-Malarial Activity of BIWS-4b on Early Infection

The
anti-malarial activity of BIWS-4b was tested in mice by the 4-day method. As
shown in Figure6, parasitemia of BIWS-4b
treated groups was significantly down (P<  .05 or
P<  .01), in a
dose-dependent manner, compared with the control group. Correspondingly,
chemosuppression was significantly elevated. Chemosuppression was up 27.84% in
mice administered oral doses of 100 mg kg−1 day−1
compared to controls. Artesunate is an effective anti-malarial drug used in the
current market. Artesunate-induced chemosuppression was 58.59% at a dose of
14 mg kg−1 day−1. Co-administration of
BIWS-4b and artesunate resulted in 89.85% chemosuppression, which is higher
than the sum of the suppression caused by BIWS-4b and artesunate individually,
indicating that BIWS-4b and artesunate acted synergistically.Figure 6
Anti-malarial effect of BIWS-4b on early
infection. After inoculating withP. yoelii, the mice were
orally treated with A: artesunate (14 mg kg−1), B: BIWS-4b (50, 100
and 200 mg kg−1), or combination of A and B for four consecutive
days. Twenty-four hours after the final administration of drug or saline,
parasitemia (%) was assessed in blood smears from tail blood of each mouse.
Each value represents the mean ± S.D. The significant
values are indicated by *P< .05 and **P < .01.


#### 3.3.2. Anti-Malarial
Activity of BIWS-4b on Residual Infection

The protective effect of
BIWS-4b against malarial infection was examined by a residual infection assay
in mice. The polysaccharides were administrated to mice followed by inoculation
withP. yoelii. Our results indicate that BIWS-4b
significantly decreased infection by P. yoelii (Figure 7(a)), which suggests that BIWS-4b enhanced the
protective ability of mice against malaria. The protective ability increased
with the time of administration, peaking on the eighth day. BIWS-4b
significantly increased chemosuppression in the dose range of
25–500 mg kg−1 compared with the control group
(P<.05 or P<.01)  (Figure 7(b)); the
data form a bell-shaped curve. At a dose of 100 mg kg−1,
chemosuppression reached its highest level of 46.21% on the eighth day.Anti-malarial effect of BIWS-4b on
residual infection: (a) the mice were given BIWS-4b at 100 mg kg−1
and 200 mg kg−1 for 4, 6, 8, or 10 consecutive days; (b) the mice
were given BIWS-4b at 25, 50, 100, 200, 400 and 500 mg kg−1 for 8
consecutive days. Twenty-four hours after the final administration of drug or
saline, the mice were inoculated with P. yoelii. Seventy-two
hours after inoculation, parasitemia (%) was assessed in blood smears from tail
blood of each mouse. Each value represents the mean ± S.D. The significant values
are indicated by *P < .05 and **P < .01.
(a)(b)


### 3.4. Immunological
Activity of BIWS-4b

#### 3.4.1. Effect on Macrophage Phagocytosis

The effect of
BIWS-4b on macrophage phagocytosis was determined using chicken red blood cells
(CRBC) in malaria-bearing mice and normal mice. As shown in Figure8, the phagocytic rate and the phagocytic index
were significantly increased compared with the control group by BIWS-4b
treatment at doses of 100 and 200 mg kg−1
(P< .01). At a dose of
100 mg kg−1 BIWS-4b, macrophage phagocytosis was enhanced to a
greater extent than at a dose of 200 mg kg−1, and the former dose
caused the most macrophage phagocytosis in malaria-bearing mice and normal mice
at the eighth day. These results are consistent with residual infection.Effect of BIWS-4b on macrophage phagocytosis
in normal mice and malaria-bearing mice: (a) phagocytosis rate in normal mice,
(b) phagocytosis index in normal mice, (c) phagocytosis rate in malaria-bearing
mice, (d) phagocytosis index in malaria-bearing mice. Each value represents the
mean± SD. Significant
differences from the negative control were evaluated using student’s
t-test: *P< .05,
**P<.01.
(a)(b)(c)(d)


#### 3.4.2. Effect on Lymphocyte
Proliferation

Splenic lymphocyte proliferation is usually followed
to evaluate a general effect on immune cells. Lymphocyte proliferation induced
by ConA reflects T-lymphocyte activity while proliferation induced by LPS
reflects B-lymphocyte activity. The effects of BIWS-4b on ConA- and LPS-induced
lymphocyte proliferation were testedin vivo by the MTT
method. As shown in Figure 9, in the presence
of ConA or LPS, BIWS-4b significantly increased lymphocyte proliferation in
malaria-bearing or normal mice compared to the control group
(P < .05). These results indicate that BIWS-4b was able to
activate both T and B cells. At a dose of 100 mg kg−1, BIWS-4b
induced higher effect on lymphocyte proliferation than that at a dose of
200 mg kg−1 and, similar to its effect on macrophage phagocytosis,
lymphocyte proliferation both in malaria-bearing mice and normal mice peaked at
the eighth day. Taken together, the data suggest that BIWS-4b can stimulate
T/B-cells to suppress the spread of malaria.Effect of BIWS-4b on lymphocyte
proliferation in normal mice and malaria-bearing mice: (a) ConA-induced
T-lymphocyte proliferation in normal mice, (b) LPS-induced B-lymphocyte
proliferation in normal mice; (c) ConA-induced T-lymphocyte proliferation in
malaria-bearing mice; (d) LPS-induced B-lymphocyte proliferation in
malaria-bearing mice. Each value represents the mean± SD. Significant
differences from the negative control were evaluated using Student’s
t-test: *P< .05,
**P<.01.
(a)(b)(c)(d)


## 3.1.
Isolation and Purification of BIWS-4b

Fruit bodies ofB.
inquinans (Fries) were treated with hot water, and extracted
polysaccharides were precipitated in ethanol. Deproteination by the Sevag
method gave an 8% yield of the polysaccharide (BIW) from dried fruit bodies.
BIW was composed of glucose, mannose and galactose in a molar ratio of
1.0 : 1.3  : 1.0, respectively. A freeze-thaw cycle of the polysaccharide
resulted in two fractions: BIWP (precipitate, 19%) and BIWS (supernatant, 63%).
Analysis of BIWP revealed glucan, which was further purified by Sephadex G-75
gel filtration chromatography to give a homogeneous
β-(1→6)-d-glucan
[12].BIWS consists of glucose, mannose
and galactose in a molar ratio of 1 :  1 : 1 and exhibits a wide molecular weight
distribution from 15 to 50 kDa. It was fractionated by gradient precipitation
in 30, 50, 70 and 90% ethanol, which resulted in the fractions BIWS-1, BIWS-2,
BIWS-3 and BIWS-4, respectively. BIWS-1, BIWS-2 and BIWS-3 showed wide
distributions on Sepharose CL-6B or Sephadex G-75 elution profiles, while
BIWS-4 gave two relatively narrow peaks on Sephadex G-75. Hence, BIWS-4 was
fractionated on a Sephadex G-75 column (1.5×  100 cm) into two
populations: BIWS-4a collected in the void elution volume (11% yield) and
BIWS-4b (72% yield) collected in the effective fractionation range as a main
portion. BIWS-4a is composed of glucose, mannose and galactose in a molar ratio
of 5.7 : 1.0 : 8.0. Because of its low yield and wide molecular weight
distribution, BIWS-4a was not studied further.


## 3.2. Analyses of BIWS-4b

BIWS-4b is a light brown powder and its total carbohydrate content was
determined to be 96% by the phenol-H2SO4 method. The UV
spectrum of BIWS-4b contained no peaks at 260 or 280 nm, and the Bradford assay
was negative, which indicated that BIWS-4b did not contain protein and nucleic
acid. A limulus amebocyte lysate (LAL) assay revealed less than 0.015 EU
(endotoxin units) mg−1, which indicates a negative result. Analysis
of sugar content showed that BIWS-4b consists of mannose (27.2%), glucose
(15.5%) and galactose (57.3%). BIWS-4b gives a narrow symmetric peak by HPSEC
(Figure 2), and its molecular weight was
estimated to be 7.4 kDa (polydispersity index, Mw/Mn: 1.35), which suggests
that BIWS-4b is a homogenous polysaccharide fraction.Figure 2
HPSEC elution profile of
BIWS-4b.The specific rotation measurement of BIWS-4b is [α]D20 –51.9° (c 0.2, H2O). The
high negative rotation suggests the dominating presence of
β-glycosidic linkages. The
FT-IR spectrum of BIWS-4b (Figure 3) shows absorption
of
β-type glycosidic linkages
at 875.5 cm−1. The other absorption bands at 3392.2 cm−1
(hydroxyl stretching vibration), 2933.3 cm−1 (C–H stretching
vibration), and 1647.0 cm−1 (bound water) were from the
corresponding sugar residues. Methylation products were sequentially acetylated
and reduced to give seven methylated alditol acetates (Figure 4). This procedure revealed that galactosyl residues
were (1→6)-linked, (1→5)-linked and (1→5,6)-linked
Galf units; glucosyl residues were (1→4)-linked and non-reducing
terminal Glcp units; mannosyl residues were (1→2)-linked, (1→6)-linked, (1→2,6)-linked and
non-reducing terminal Manp units. The ratios of each residue
are listed in Table 1.Table 1
Methylation analysis of BIWS-4b.
Partially methylated glycitolMolar ratioaRetention time (min)Linkage type1,4,5-Tri-acetyl-2,3,6-tri-O-methyl galactitol4.617.95→5)-Galf-(1→1,4,5,6-Tetra-acetyl-2,3-di-O-methyl galactitol3.117.88→5,6)-Galf-(1→1,4,6-Tri-acetyl-2,3,5-tri-O-methyl galactitol7.618.94→6)-Galf-(1→1,4,5-Tri-acetyl-2,3,6-tri-O-methyl glucitol2.218.32→4)-Glcp-(1→1,5-Di-acetyl-2,3,4,6-tetra-O-methyl glucitol2.617.11Glcp-(1→1,2,5-Tri-acetyl-3,4,6-tri-O-methyl mannitol2.417.84→2)-Manp-(1→1,5-Tri-acetyl-2,3,4,6-tri-O-methyl mannitol1.017.06Manp-(1→1,2,5,6-Tetra-acetyl-3,4-di-O-methyl mannitol1.218.82→2,6)-Manp-(1→1,5,6-Tri-acetyl-2,3,4-tri-O-methyl mannitol2.918.72→6)-Manp-(1→aRelative molar ratio, calculated from the ratio of peak area.Figure 3
FT-IR spectra: (a) BIWS-4b and (b) partially
methylated BIWS-4b.Figure 4
GC profile of BIWS-4b in
methylation analysis.The
structural features of BIWS-4b were further analyzed by13C NMR;
assignments of carbon atom signals are listed in
Table 2 and Figure 5.
According to NMR data in the literatures [22–28], the
anomeric carbon signals at
δ 107.73, 107.03 and 106.92
can be attributed to C-1 of three different linkages of
β-Galf
residues;
δ 102.43 and 102.19
correspond to C-1 of
β-Glcp and
δ~101.19 to 97.24
correspond to C-1 of
α-Manp.
These results were consistent with those of methylation analysis. Combining the
results from methylation and NMR analyses, we deduced that BIWS-4b was mainly
composed of (1→6)-linked, (1→5)-linked, and (1→5,6)-linked
β-Galf
units, as well as (1→4)-linked, non-reducing
terminal
β-Glcp
units, and (1→2)-linked, (1→6)-linked, (1→2,6)-linked and
non-reducing terminal
α-Manp
units.Table 2
13C NMR chemical shifts of BIWS-4b in D2O.
Residueδ (ppm)C-1C-2C-3C-4C-5C-6→5)-β-Galf-(1→107.7380.9376.8181.1875.4762.73→5,6)-β-Galf-(1→107.0381.4576.4082.5074.2068.01→6)-β-Galf-(1→106.9282.0076.6583.9869.8969.70→4)-β-Glcp-(1→102.4373.1874.3079.0974.8360.64β-Glcp-(1→102.1972.9674.2069.1275.8360.32α-Manp-(1→101.1969.1269.5466.5672.4060.10→2)-α-Manp-(1→99.4577.2568.7666.6772.4060.10→6)-α-Manp-(1→98.3769.1269.5466.5871.9564.89→2,6)-α-Manp-(1→97.2477.6269.4066.6771.9564.92Figure 5
13C NMR spectrum of BIWS-4b. Solvent:
D2O, temperature: 20°C.


## 3.3. Anti-Malarial Activity of
BIWS-4b

### 3.3.1.
Anti-Malarial Activity of BIWS-4b on Early Infection

The
anti-malarial activity of BIWS-4b was tested in mice by the 4-day method. As
shown in Figure6, parasitemia of BIWS-4b
treated groups was significantly down (P<  .05 or
P<  .01), in a
dose-dependent manner, compared with the control group. Correspondingly,
chemosuppression was significantly elevated. Chemosuppression was up 27.84% in
mice administered oral doses of 100 mg kg−1 day−1
compared to controls. Artesunate is an effective anti-malarial drug used in the
current market. Artesunate-induced chemosuppression was 58.59% at a dose of
14 mg kg−1 day−1. Co-administration of
BIWS-4b and artesunate resulted in 89.85% chemosuppression, which is higher
than the sum of the suppression caused by BIWS-4b and artesunate individually,
indicating that BIWS-4b and artesunate acted synergistically.Figure 6
Anti-malarial effect of BIWS-4b on early
infection. After inoculating withP. yoelii, the mice were
orally treated with A: artesunate (14 mg kg−1), B: BIWS-4b (50, 100
and 200 mg kg−1), or combination of A and B for four consecutive
days. Twenty-four hours after the final administration of drug or saline,
parasitemia (%) was assessed in blood smears from tail blood of each mouse.
Each value represents the mean ± S.D. The significant
values are indicated by *P< .05 and **P < .01.


### 3.3.2. Anti-Malarial
Activity of BIWS-4b on Residual Infection

The protective effect of
BIWS-4b against malarial infection was examined by a residual infection assay
in mice. The polysaccharides were administrated to mice followed by inoculation
withP. yoelii. Our results indicate that BIWS-4b
significantly decreased infection by P. yoelii (Figure 7(a)), which suggests that BIWS-4b enhanced the
protective ability of mice against malaria. The protective ability increased
with the time of administration, peaking on the eighth day. BIWS-4b
significantly increased chemosuppression in the dose range of
25–500 mg kg−1 compared with the control group
(P<.05 or P<.01)  (Figure 7(b)); the
data form a bell-shaped curve. At a dose of 100 mg kg−1,
chemosuppression reached its highest level of 46.21% on the eighth day.Anti-malarial effect of BIWS-4b on
residual infection: (a) the mice were given BIWS-4b at 100 mg kg−1
and 200 mg kg−1 for 4, 6, 8, or 10 consecutive days; (b) the mice
were given BIWS-4b at 25, 50, 100, 200, 400 and 500 mg kg−1 for 8
consecutive days. Twenty-four hours after the final administration of drug or
saline, the mice were inoculated with P. yoelii. Seventy-two
hours after inoculation, parasitemia (%) was assessed in blood smears from tail
blood of each mouse. Each value represents the mean ± S.D. The significant values
are indicated by *P < .05 and **P < .01.
(a)(b)


## 3.3.1.
Anti-Malarial Activity of BIWS-4b on Early Infection

The
anti-malarial activity of BIWS-4b was tested in mice by the 4-day method. As
shown in Figure6, parasitemia of BIWS-4b
treated groups was significantly down (P<  .05 or
P<  .01), in a
dose-dependent manner, compared with the control group. Correspondingly,
chemosuppression was significantly elevated. Chemosuppression was up 27.84% in
mice administered oral doses of 100 mg kg−1 day−1
compared to controls. Artesunate is an effective anti-malarial drug used in the
current market. Artesunate-induced chemosuppression was 58.59% at a dose of
14 mg kg−1 day−1. Co-administration of
BIWS-4b and artesunate resulted in 89.85% chemosuppression, which is higher
than the sum of the suppression caused by BIWS-4b and artesunate individually,
indicating that BIWS-4b and artesunate acted synergistically.Figure 6
Anti-malarial effect of BIWS-4b on early
infection. After inoculating withP. yoelii, the mice were
orally treated with A: artesunate (14 mg kg−1), B: BIWS-4b (50, 100
and 200 mg kg−1), or combination of A and B for four consecutive
days. Twenty-four hours after the final administration of drug or saline,
parasitemia (%) was assessed in blood smears from tail blood of each mouse.
Each value represents the mean ± S.D. The significant
values are indicated by *P< .05 and **P < .01.


## 3.3.2. Anti-Malarial
Activity of BIWS-4b on Residual Infection

The protective effect of
BIWS-4b against malarial infection was examined by a residual infection assay
in mice. The polysaccharides were administrated to mice followed by inoculation
withP. yoelii. Our results indicate that BIWS-4b
significantly decreased infection by P. yoelii (Figure 7(a)), which suggests that BIWS-4b enhanced the
protective ability of mice against malaria. The protective ability increased
with the time of administration, peaking on the eighth day. BIWS-4b
significantly increased chemosuppression in the dose range of
25–500 mg kg−1 compared with the control group
(P<.05 or P<.01)  (Figure 7(b)); the
data form a bell-shaped curve. At a dose of 100 mg kg−1,
chemosuppression reached its highest level of 46.21% on the eighth day.Anti-malarial effect of BIWS-4b on
residual infection: (a) the mice were given BIWS-4b at 100 mg kg−1
and 200 mg kg−1 for 4, 6, 8, or 10 consecutive days; (b) the mice
were given BIWS-4b at 25, 50, 100, 200, 400 and 500 mg kg−1 for 8
consecutive days. Twenty-four hours after the final administration of drug or
saline, the mice were inoculated with P. yoelii. Seventy-two
hours after inoculation, parasitemia (%) was assessed in blood smears from tail
blood of each mouse. Each value represents the mean ± S.D. The significant values
are indicated by *P < .05 and **P < .01.
(a)(b)


## 3.4. Immunological
Activity of BIWS-4b

### 3.4.1. Effect on Macrophage Phagocytosis

The effect of
BIWS-4b on macrophage phagocytosis was determined using chicken red blood cells
(CRBC) in malaria-bearing mice and normal mice. As shown in Figure8, the phagocytic rate and the phagocytic index
were significantly increased compared with the control group by BIWS-4b
treatment at doses of 100 and 200 mg kg−1
(P< .01). At a dose of
100 mg kg−1 BIWS-4b, macrophage phagocytosis was enhanced to a
greater extent than at a dose of 200 mg kg−1, and the former dose
caused the most macrophage phagocytosis in malaria-bearing mice and normal mice
at the eighth day. These results are consistent with residual infection.Effect of BIWS-4b on macrophage phagocytosis
in normal mice and malaria-bearing mice: (a) phagocytosis rate in normal mice,
(b) phagocytosis index in normal mice, (c) phagocytosis rate in malaria-bearing
mice, (d) phagocytosis index in malaria-bearing mice. Each value represents the
mean± SD. Significant
differences from the negative control were evaluated using student’s
t-test: *P< .05,
**P<.01.
(a)(b)(c)(d)


### 3.4.2. Effect on Lymphocyte
Proliferation

Splenic lymphocyte proliferation is usually followed
to evaluate a general effect on immune cells. Lymphocyte proliferation induced
by ConA reflects T-lymphocyte activity while proliferation induced by LPS
reflects B-lymphocyte activity. The effects of BIWS-4b on ConA- and LPS-induced
lymphocyte proliferation were testedin vivo by the MTT
method. As shown in Figure 9, in the presence
of ConA or LPS, BIWS-4b significantly increased lymphocyte proliferation in
malaria-bearing or normal mice compared to the control group
(P < .05). These results indicate that BIWS-4b was able to
activate both T and B cells. At a dose of 100 mg kg−1, BIWS-4b
induced higher effect on lymphocyte proliferation than that at a dose of
200 mg kg−1 and, similar to its effect on macrophage phagocytosis,
lymphocyte proliferation both in malaria-bearing mice and normal mice peaked at
the eighth day. Taken together, the data suggest that BIWS-4b can stimulate
T/B-cells to suppress the spread of malaria.Effect of BIWS-4b on lymphocyte
proliferation in normal mice and malaria-bearing mice: (a) ConA-induced
T-lymphocyte proliferation in normal mice, (b) LPS-induced B-lymphocyte
proliferation in normal mice; (c) ConA-induced T-lymphocyte proliferation in
malaria-bearing mice; (d) LPS-induced B-lymphocyte proliferation in
malaria-bearing mice. Each value represents the mean± SD. Significant
differences from the negative control were evaluated using Student’s
t-test: *P< .05,
**P<.01.
(a)(b)(c)(d)


## 3.4.1. Effect on Macrophage Phagocytosis

The effect of
BIWS-4b on macrophage phagocytosis was determined using chicken red blood cells
(CRBC) in malaria-bearing mice and normal mice. As shown in Figure8, the phagocytic rate and the phagocytic index
were significantly increased compared with the control group by BIWS-4b
treatment at doses of 100 and 200 mg kg−1
(P< .01). At a dose of
100 mg kg−1 BIWS-4b, macrophage phagocytosis was enhanced to a
greater extent than at a dose of 200 mg kg−1, and the former dose
caused the most macrophage phagocytosis in malaria-bearing mice and normal mice
at the eighth day. These results are consistent with residual infection.Effect of BIWS-4b on macrophage phagocytosis
in normal mice and malaria-bearing mice: (a) phagocytosis rate in normal mice,
(b) phagocytosis index in normal mice, (c) phagocytosis rate in malaria-bearing
mice, (d) phagocytosis index in malaria-bearing mice. Each value represents the
mean± SD. Significant
differences from the negative control were evaluated using student’s
t-test: *P< .05,
**P<.01.
(a)(b)(c)(d)


## 3.4.2. Effect on Lymphocyte
Proliferation

Splenic lymphocyte proliferation is usually followed
to evaluate a general effect on immune cells. Lymphocyte proliferation induced
by ConA reflects T-lymphocyte activity while proliferation induced by LPS
reflects B-lymphocyte activity. The effects of BIWS-4b on ConA- and LPS-induced
lymphocyte proliferation were testedin vivo by the MTT
method. As shown in Figure 9, in the presence
of ConA or LPS, BIWS-4b significantly increased lymphocyte proliferation in
malaria-bearing or normal mice compared to the control group
(P < .05). These results indicate that BIWS-4b was able to
activate both T and B cells. At a dose of 100 mg kg−1, BIWS-4b
induced higher effect on lymphocyte proliferation than that at a dose of
200 mg kg−1 and, similar to its effect on macrophage phagocytosis,
lymphocyte proliferation both in malaria-bearing mice and normal mice peaked at
the eighth day. Taken together, the data suggest that BIWS-4b can stimulate
T/B-cells to suppress the spread of malaria.Effect of BIWS-4b on lymphocyte
proliferation in normal mice and malaria-bearing mice: (a) ConA-induced
T-lymphocyte proliferation in normal mice, (b) LPS-induced B-lymphocyte
proliferation in normal mice; (c) ConA-induced T-lymphocyte proliferation in
malaria-bearing mice; (d) LPS-induced B-lymphocyte proliferation in
malaria-bearing mice. Each value represents the mean± SD. Significant
differences from the negative control were evaluated using Student’s
t-test: *P< .05,
**P<.01.
(a)(b)(c)(d)


## 4. Discussion

Polysaccharides from fungi often possess immunological activities that can
protect the body from microbial and parasitic attack [29–31]. Although some polysaccharides from
ascomycetous lichens are reported to be composed of galactofuranose,
glucopyranose and mannopyranose residues [25, 27, 28, 32–37], little work about possible anti-malarial
activity has been performed. Lentinan (1,3-β-d-glucan) has
recently been investigated for its anti-malarial activity using the P.
yoelii blood-stage infection model and found to possess prophylactic
potential for the treatment of malaria via immunostimulation
[9]. In this article, we isolated, for the first
time, a heteropolysaccharide fraction (BIWS-4b) containing
α-d-Manp,
β-d-Glcp and
β-d-Galf from the non-lichenized edible
mushroom B. inquinans (Fries). Considering the complex
structure of BIWS-4b, we supposed it may have potential biological activities.
Therefore, BIWS-4b was evaluated for its complementary therapy and prophylactic
activities against malaria as well as its immunostimulating activity. The
results indicated that BIWS-4b had markedly suppressive activity on early or
residual infection. Thus, it is possible that BIWS-4b could be used as a
complementary drug or protective agent against malaria.Artesunate is an
effective drug currently used to cure malarial infection. The endoperoxide
moiety of its molecular structure can oxidize parasitic membranes and react
with heme to form a cytotoxic carbon-centered radical intermediate that further
reacts with susceptible groups within parasite enzymes and lipids [38–40]. However, artesunate has some negative side
effects including interfering with some aspects of the host immune system,
resulting in the difficulty of establishing a long-lasting protective immunity
against malaria [41]. Therefore,
artesunate-based combination therapy was recommended by the World Health
Organization (WHO) as a new prospective paradigm [42]. Lymphocytes are the key effecter cells of the mammalian immune
system and stimulating macrophages is another way to enhance immunological
activity [43, 44].
BIWS-4b significantly enhanced spleen lymphocyte proliferation upon stimulation
by ConA or LPS and augmented macrophage phagocytosis in malarial-bearing or
normal mice. In our experiments, it was observed that both immunological
enhancement of BIWS-4b and its protection against malarial infection peaked at
100 mg kg−1 on Day 8, which implied that its anti-malarial activity
might be intermediated by enhancing host immune competence. In combination with
artesunate, BIWS-4b was able to recover immune system functionality that was
previously suppressed by artesunate in mice. The synergistic effect of BIWS-4b
and artesunate may be utilized to enhance and restore artesunate-suppressed
immune function. Similar results were observed for lentinan [9].The prophylactic activity against malaria
and the immunological enhancement of BIWS-4b exhibited a bell-shaped curve. One
interpretation of this effect is that signal transduction mechanisms underlying
the action of lymphocytes and macrophages might be regulated downward by
exposure to a higher dosage of BIWS-4b [45]. An
alternative interpretation is that BIWS-4b has not only effects which promote
the immune system, but also those which inhibit their system, and the promoting
mechanisms might be masked by its inhibitory effects at a higher dosage [46]. Similar results have been observed in some
polysaccharides when used as complementary drugs for anti-cancer treatments
[47–50], such as
the tumor growth inhibition of a Cordyceps sinensis
polysaccharide [47] and the TNF-α level increase by an
Aloe vera polysaccharide [48].In conclusion, a water-soluble heteropolysaccharide
BIWS-4b was purified from fruit bodies ofB. inquinans
(Fries). It was found to contain (1→6)-, (1→5)- and (1→5,6)-linked
β-Galf
units, as well as (1→4)-linked non-reducing
terminal
β-Glcp
units, and (1→2)-linked, (1→6)-linked, (1→2,6)-linked and
non-reducing terminal
α-Manp
units. BIWS-4b was shown to possess marked suppressive activity against early
infection of malaria by the 4-day method in mice. Residual infection and
immunological assays indicated that BIWS-4b had significant prophylactic
activity against malaria by stimulating the immune system. Moreover, BIWS-4b
increased anti-malarial activity of artesunate at early infection by oral
administration in combination. The deduced anti-malarial action of BIWS-4b was
shown in Figure 10. Therefore, BIWS-4b could be
considered a potential adjuvant of anti-malarial drugs.Figure 10
The deduced anti-malarial action of BIWS-4b: when
infected with malaria, the host generates a protective immune response,
including cellular and humoral immunity. BIWS-4b can significantly enhance the
immune competence of the host and exhibits remarkable prophylactic activity
against malaria. In combination with artesunate, BIWS-4b can restore the
artesunate-suppressed immune function and enhance the anti-malarial activity of
artesunate.


## Funding

National
Natural Science Foundation of China (no. 30570417, 30770489) and the Natural
Science Foundation of Jilin Province (no. 20070710).



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*Source: 973460-2011-05-03.xml*