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

A water-soluble polysaccharide (BIWS-4b) was purified from the fruit bodies of Bulgaria 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.


Introduction
Many fungi are used as traditional medicines in the treatment of various human diseases such as hepatitis, hypertension, hypercholesterolemia and gastric cancer [1][2][3][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][6][7][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 ascomycete Bulgaria 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.

Materials and Chemicals. Fruit bodies of B. 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.

Isolation and
Purification of BIWS-4b. The isolation of polysaccharides from fruit bodies of B. 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 × 10 3 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.  [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.
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.
The 13 C 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 D 2 O (99.8%, 0.5 ml), freeze-dried, redissolved in D 2 O (0.5 ml) and centrifuged to remove excess sample. The experiment was recorded using standard Bruker software.

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 PW XL 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 CHCl 3 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 CF 3 COOH (2 M, 1 ml) for 6 h at 100 • C. Partially methylated sugars in the hydrolysate were reduced by NaBH 4 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.

Preliminary Anti-Malaria Testing In Vivo
The Plasmodium 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 ×10 7 ml −1 P. yoelii parasitized erythrocytes from a donor mouse, which was used to infect mice intraperitoneally.

Suppressive Activity on Early Infection (4-Day Test).
Suppression of early infection was evaluated using a 4day 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 where A is the average parasitemia of the negative control group and B is the average parasitemia of the test group.

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. Evidence-Based Complementary and Alternative Medicine 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.

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% CO 2 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.

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 Tris 2 NH 4 Cl (0.16 M, Tris 2 NH 4 Cl, 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 flatbottom microtiter plates at 5 ×10 6 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% CO 2 . 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.

Isolation and Purification of BIWS-4b.
Fruit bodies of B. 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].

Analyses of BIWS-4b.
BIWS-4b is a light brown powder and its total carbohydrate content was determined to be 96% by the phenol-H 2 SO 4 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. (1 → 6)-linked, (1 → 2,6)-linked and non-reducing terminal Manp units. The ratios of each residue are listed in Table 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 Figure 6, 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.

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 with P. 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 bellshaped curve. At a dose of 100 mg kg −1 , chemosuppression reached its highest level of 46.21% on the eighth day. Evidence-Based Complementary and Alternative Medicine Chemical shift (ppm)

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 Figure 8, 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 malariabearing mice and normal mice at the eighth day. These results are consistent with residual infection.

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 tested in vivo by the MTT method. As shown in Figure 9, in the presence of ConA or LPS, BIWS-4b significantly increased lymphocyte proliferation in malariabearing 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.

Discussion
Polysaccharides from fungi often possess immunological activities that can protect the body from microbial and parasitic attack [29][30][31]. Although some polysaccharides from ascomycetous lichens are reported to be composed of galactofuranose, glucopyranose and mannopyranose residues [25,27,28,[32][33][34][35][36][37], little work about possible antimalarial activity has been performed. Lentinan (1,3-β-dglucan) 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][39][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  Figure 8: 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.
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 bellshaped 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][48][49][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 of B. 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 nonreducing 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. Malaria killing 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).