Effect of the Molecular Mass of Tremella Polysaccharides on Accelerated Recovery from Cyclophosphamide-Induced Leucopenia in Rats

The body of tremella were decocted with water, and hydrolyzed with 0.1 mol/L hydrochloric acid for different times, giving tremella polysaccharides with six molecular mass values. The structures of all the tremella polysaccharides had non-reducing terminals of β-D-pyranglucuronide, the backbone was composed of (1→3)-linked β-D-manno-pyranoside, and the side chain composed of (1→6)-linked β-D-xylopyranoside was attached to the C2 of the backbone mannopyranoside. Immunomodulatory effect studies indicated that tremella polysaccharides increased the counts of leukocytes in the peripheral blood which were significantly lowered by cyclophosphamide, and the lower the molecular mass of the tremella polysaccharide, the better this effect was.


Introduction
Cancer is one of the leading causes of death in the world. Chemotherapeutics are often used to inhibit the proliferation of cancer cells. Cyclophosphamide (Cy) is the most widely used alkylating agent in cancer chemotherapy to date. The anti-tumor effect of Cy is proportional to the dose of Cy administered, often resulting in unwanted immunosuppressive and cytotoxic effects [1]. Chemotherapy-induced leukopenia leads to significant morbidity and mortality, a major limiting factor in clinical chemotherapy without efficacious remedies. In Traditional Chinese Medicine, co-administration of immunomodulatory agents and chemotherapy drugs is typically used to improve the immunity potential. In many oriental countries, several immunoceuticals composed of polysaccharides are widely used, such as lentinan (LT), schizophyllan and krestin. Those immunomodulatory agents often act by inducing lymphocyte proliferation and cytokine production, and they have protective effects toward the hematopoietic function of bone marrow and immune organs [2].
The body of tremella (Tremella fuciformis) is a popular food and herbal medicine, widely used in Asian countries as a tonic [3]. Tremella polysaccharide (TP) has received extensive attention. TP consist of a (1→3)-mannan backbone with small xylose-, glucose-, arabinose-, fucose-and glucuronic acid-containing side chains [4]. It was reported that TP exerted an anti-aging effect by increasing the levels of superoxide dismutase (SOD), a key antioxidant enzyme in brain and liver cells [5], and other pharmacological activities, including cytokine-stimulating, anti-tumor, anti-diabetic, antiinflammatory, vascular-stimulating, cholesterol-lowering, antiallergic and hepatoprotective effects have been reported [6].
In this paper, we investigated the structural characterisation of TPs with six different molecular mass values, and their immunomodulatory effect against cyclophosphamide-induced leucopenia in rats. We found that TP showed the immunomodulatory effects against cyclophosphamide-induced leucopenia in rats in a dose-dependent manner, and the lower the molecular mass of TP was, the better was the activity.
As shown in Table 2, glucuronic acid was reduced to be glucose before methylation, so 2,3,4,6-Me 4 -D-Glc is the methylation product of glucuronic acid. The structures of TP-1, TP-2, TP-3, TP-4, TP-5 and TP-6 had non-reducing terminals of β-D-pyranglucuronide, the backbone was composed of (1→3)-linked β-D-mannopyranoside, and the side chain, composed of (1→6)-linked β-D-xylopyranoside, was attached through C 2 of the backbone mannosyl residues. The structure of TP-1 to TP-6 (shown in Figure 2) is consistent with tremella polysaccharide, which means the tremella polysaccharide has repeating units which can be prepared by acid hydrolysis. Because the glycosidic bond connected with glucuronic acid was hard to hydrolyze by acid hydrolysis [8], the side chains of TP contained glucuronic acid that was not hydrolyzed.  The TP didn't showed immunomodulatory effects against cyclophosphamide-induced leucopenia in rats by intraperitoneal injection (data not shown). This is because the molecular mass of TP was high, the viscosity was too high to dissolve it properly. As shown in Table 3, the administration of TP-1 to TP-6, respectively, once a day increased the counts of leukocytes in the peripaeral blood which were significantly lowered by Cy in a dose-dependent manner. TP-6 showed the best immunomodulatory effect against cyclophosphamide-induced leucopenia in rats (in Table 3), the results of both low dose and high dose group were better than matrine group from day 3 to day 9. By day 9, the leukocyte numbers of TP-6 group with 200 mg/kg/d had returned to the level of normal rats. Table 3. Accelerated recovery from cyclophosphamide-induced leucopenia in rats ( X ± SD, n = 10). Cy reduces the bone marrow cellularity and the number of leucocytes in peripheral blood and the thymidine incorporation in bone marrow cells [9]. TP-6 could increase the counts of leukocytes in the peripheral blood which were significantly lowered by Cy, probably because it could enhance the haemopoiesis of bone marrow. The results suggested that TP-6 could increase the counts of bone marrow cellularity and the DNA in bone marrow cells.
Previously, we found that after oral administration of TP only trace amounts (about 0.4%) was passed through the gastrointestinal tract into the blood and absorbed by organs [10]. Among TP-1, TP-2, TP-3, TP-4, TP-5 and TP-6, the results of the physicochemical properties, monosaccharide compositions and structure analysis are similar, but the molecular mass is different. From the results in Table 3, we inferred that the lower the molecular mass of TP, the activity was better. Perhaps this is because the permeability is proportional to the molecular mass, so low molecular mass TP could easily pass through cell membranes, be absorbed by organs and show the effects.

The Preparation of Six Kinds of Molecular Mass Tremella Polysaccharide
Body of tremella was purchased from a local market in Changchun City (China). Tremella (1 Kg) was cut into small pieces and decocted three times with water (50 L), then the aqueous solutions were combined into one portion, and evaporated. The aqueous extract was hydrolyzed with 0.1 mol/L hydrochloric acid at 90 °C for 0.5 h, 1.5 h, 2 h, 2.5 h, 3 h and 3.5 h to obtained six hydrolysates. After hydrolysis, 20% NaOH was added to the six hydrolysates to adjuated the pH to 7.0, then they were centrifuged at 3000 rpm for 10 min, and the supernatants forced through a Cellulose Superfiltration System to give the high molecular weight fractions, which were then lyophilised. The six dry samples were separately applied into a Sephadex G-150 column (2.5 × 90 cm, from Sigma Chemical Co.), and eluted with water (800 mL). Major fractions were collected according to their elution patterns. Six samples (named TP-1, TP-2, TP-3, TP-4, TP-5 and TP-6) were obtained (Figure 1).

IR Study
The FT-IR spectrum was acquired using a Bruker (Germany) Vertex 70 FTIR instrument. The sample was pressed into KBr pellets and the spectra were recorded in a transmittance mode over a wavelength range between 4,000 and 400 cm −1 .