Modification of Low Molecular Weight Polysaccharides from Tremella Fuciformis and Their Antioxidant Activity in Vitro

In this study, sulfated low molecular-weight Tremella fuciformis polysaccharides (SLTP) with different sulfate contents were synthesized and their antioxidant activities, including superoxide anion radical, 1,1-diphenyl-2-picryl-hydrazyl (DPPH) radical and hydroxyl radical scavenging activities were investigated. The results indicated that, compared to natural Tremella fuciformis polysaccharide (TP) and low molecular weight Tremella fuciformis polysaccharide (LTP), sulfated LTP (SLTP) exhibited stronger scavenging activity towards superoxide anion, DPPH and hydroxyl radicals. In all the cases the effect was found to be dose dependent. The scavenging activity of SLTP was found to be in parallel with the degree of sulfation of SLTP.


Introduction
Tremella fuciformis is a common nutritional food in China. It is also a traditional Chinese medicine regarded as a tonic for "weakness and aging". Such dietary and medicinal properties are assumed to be due solely to the polysaccharides produced by the Tremella species. TP is usually extracted from Tremella fuciformisis fruit bodies or fermentation mycelia. Its average molecular weight is in the range of 12~50 kD. TP has a mannose backbone and side chains containing glucosyl, fucosyl xylosyl, and glucuronic acid residues [1]. TP has been found to show several biological activities such as anti-inflammation, anti-cancer, anti-mutation, reducing hypertension, reducing blood sugar and immunity improving properties [2]. The molecular weight of polysaccharides has significant effect on their biological activities. High molecular weight polysaccharides are not conducive to easy crossing of cell-membranes, and exert their biological activities with difficulty. Conceptually, degradation of high molecular weight polysaccharides into low molecular weight ones might improve their biological activities noticeably and therefore, preparation of low molecular weight polysaccharides becomes an interesting method for preparation of new drugs. Chemical modification of polysaccharides provides an opportunity to obtain new pharmacological agents with possible therapeutic uses [3]. It has been reported that sulfate groups of polysaccharides probably contribute to their biological activities [4].
Reactive oxygen species (ROS), capable of causing damage to DNA, have been associated with carcinogenesis, coronary heart disease and many other health problems related to aging [5,6]. Thus, it is essential to develop and utilize effective and natural antioxidants so that they can protect the human body against free radicals and retard the progress of many chronic diseases [7].
Until now, studies on TP are mainly focused on the extraction and pharmacological activity. However, little information is available concerning the degradation of TP and the modification of LTP. In the present study, sulfation of LTP was done under various conditions to obtain different SLTPs such as SLTPI, SLTPII and SLTPIII and their radical scavenging activity has been compared with the naturally existing TP and LTP and the results obtained are presented.

Materials
TP (Average molecular weight is 32 kD) was prepared as described by Wu et al. [8]. DPPH, nicotinamide adenine dinucleotide-reduced (NADH), thiobarbituric acid (TBA), trichloroacetic acid (TCA), nitro blue tetrazolium (NBT), and Vitamin C (Vc) were purchased from Sigma Chemical Co. All others chemicals and reagents were of analytical grade.

Preparation of LTP
LTP was obtained by hydrolyzing TP in the following way: the ratio of TP/solvent (w/v) is 1:50, the concentration of HCl is 0.7 M, 80 °C of water bath for 2 h. The average molecular weight of LTP was 1.2 kD. The molecular weight was determined with a Waters gel permeation chromatography (GPC) [9].

Modification of LTP
SLTP was synthesized by the chlorosulfonic acid-pyridine method [10]. The sulfation agent, pyridine-SO 3 complex, was obtained by dropping chlorosulfonic acid (HClSO 3, 2 ml) into pyridine (10 ml) in an ice-water bath. Dry LTP (0.2 g) was added to formamide (FA, 20 ml), and the mixture was stirred at different temperatures (60, 80, 100 °C) for 30 min to disperse it evenly. Then pyridine-SO 3 complex was added. After 2 h, the mixture was cooled to room temperature, neutralized with 1 M NaOH solution, dialyzed against tap water for 72 h, and precipitated with 95 % ethanol for 24 h. The precipitate was filtered off and washed three times with ethanol, then lyophilized to give SLTP. Three SLTPs with different sulfate content were obtained by changing the reaction temperature with fixed molar ratio of LTP/pyridine-SO 3 complex and reaction time. Sulfate content was determined by using the traditional method of barium chloride-gelatin [11] to evaluate degree of sulphatation (DS). (1)

DPPH Radical Scavenging Activity of SLTP, LTP and TP
The DPPH radical scavenging activity was tested by the method of Li et al. [12]. A 0.1 mM solution of DPPH in ethanol was prepared and this solution (1 ml) was added to sample (10-50 µg/ml, 2 ml) in water. After 30 min, absorbance was measured at 517 nm. Decreased absorbance of the reaction mixture indicated higher DPPH radical scavenging activity. The DPPH concentration (C DPPH ) in the reaction medium was calculated from the following calibration curve, determined by linear regression (R 2 = 0.9035): Absorbance =-2.9535 × C DPPH .
The scavenging activity of DPPH radical was calculated using the following equation:

Superoxide Anion radical Scavenging Activity of SLTP, LTP and TP
The superoxide radical scavenging activity was assessed by the method of Robak et al. [14]. The reaction mixture, containing sample (10-50 µg/ml), Tris-HCl (16 mM, pH 8.0), NADH (338 µM), NBT (72 µM), and PMS (30 µM), was incubated at room temperature for 5 min and the absorbance was measured at 560 nm against a blank. The scavenging activity of superoxide anion radical was calculated using the following equation:

Statistical Analysis
Data were presented as means±standard deviations (SD) of three determinations and followed by Student's t test. Differences were considered to be statistically significant if P < 0.05.

Modification of LTP
Sulfated LTP were obtained by the chlorosulfonic acid/pyridine method. Three sulfated derivatives named SLTPI, SLTPII and SLTPIII were synthesized at different temperatures. The DS and water solubility are shown in Table 1. With the elevation of the temperature from 60 to 100 °C, the DS of the corresponding derivatives increased from 0.41 to 1.02. This indicated that with the elevation of temperature the DS increased. Water solubility was SLTP > LTP > TP. It showed that the degradation and sulfation improved the water solubility of TP.
The main monosaccharide unit in LTP is mannose. The axial -OH is more likely to be sulfated than equatorial -OH [15]. LTP has few OH groups bound to the C-1 atom. This makes the -OH group on C-2 easy to be sulfated. It has been reported that reactivity of polysaccharide hydroxyl group was C-6 > C-2 > C-4. The low sulfation at C-4 can be attributed to the steric hindrance [16].  Results are presented as mean ± SD (n = 3). Figure 2 shows the DPPH radical scavenging activity of TP, LTP, SLTP and Vc. The scavenging activity increased with increasing concentration from 10 to 50 µg/ml and sulfate content from 0.41 to 1.12. Among all the samples and Vc, SLTPIII exhibited the strongest scavenging activity against DPPH radical. Moreover, the three SLTPs showed higher scavenging activity than Vc (P < 0.05). TP showed the lowest scavenging activity. At the concentration of 10 µg/mL, the scavenging activity was 38.59 % for SLTPIII, while the concentration of SLTPIII was 50 µg/ml, a scavenging activity of 53.8 % was obtained. Results are presented as mean ± SD (n = 3).

Figure 2. Scavenging activity of natural TP, its derivatives and Vitamin C against DPPH radicals.
Values were means ± SD (n = 3).

Hydroxyl radical scavenging activity of SLTP, LTP and TP
Hydroxyl radical and its subsequent radicals are the most harmful ROS and they are responsible for the oxidative injury of biomolecules [17]. The scavenging activity of all samples and Vc are shown in Figure 3. The results suggested that SLTP, possessed more pronounced hydroxyl radical scavenging activity than LTP, TP and Vc (P < 0.05), and the scavenging activity increased with increasing the concentration. The scavenging activity of SLTPIII was obviously higher than other polysaccharides, and the highest scavenging activity was 65.9 %. The scavenging activity of SLTPIII at 10 µg/ml was found to be 36.1 %. The same scavenging activity could be obtained for LTP at 50 µg/ml. Vc showed the lowest scavenging activity against hydroxyl radical among all samples tested.

Superoxide anion radical scavenging activity of SLTP, LTP and TP
Among all the reactive oxygen species, superoxide anion radical is generated firstly. Although it is a relatively weak oxidant, it can decompose to form stronger reactive oxidative species, such as single oxygen and hydroxyl radicals [17]. Further, superoxide anion radicals are also known to indirectly initiate lipid peroxidation as a result of H 2 O 2 formation, creating precursors of hydroxyl radicals [18]. So, scavenging activity of superoxide anion radical is an important index for antioxidants.   SLTP exhibited a moderate superoxide anion radical scavenging activity than Vc, LTP and TP (P < 0.05), and the scavenging activity increased with increasing the concentration. The scavenging activity of SLTPIII at 10 µg/ml was found to be 36.4 %. That of SLTPII at 20 µg/ml was 37.2 %, while a similar value was obtained for LTP at 50 µg/ml. The percentage inhibition of superoxide generation for SLTPIII at 50 µg/ml was found to be 45 %. The value at this concentration for SLTPII was 40 % (Figure 4). Results are presented as mean ± SD (n = 3).  The results of antioxidant assay indicated that SLTP, LTP and TP scavenged free radicals in a concentration-dependent manner. When the concentration was 50 µg/ml, the antioxidant activity became the highest. The antioxidant activity of LTP was higher than those of TP, but lower than those of SLTP. Among three SLTP samples, the highest sulfated content SLTP had the strongest antioxidant activity. We also studied the antioxidant activity of Vc using the above-mentioned model. SLTP showed stronger scavenging activity against free radical than Vc (P < 0.05). It suggested that introduction of sulfonic group into LTP could afford more effective protection against damage. The results in this study suggested that SLTP, possessing pronounced free radical scavenging activity, could be of considerable preventive significance to some life-threatening health problems such as cancer, atherogenesis and Alzheimer's disease, which pathologically initiated by the presence of free radicals leading to the inevitable peroxidation of important biomolecules [17].
One of the mechanisms involving in antioxidant activity is the ability of a molecule to donate hydrogen atom to radical and the propensity of hydrogen donation is the critical factor affecting free radical scavenging activity [19]. The SLTP appeared to function as good hydrogen atom donors and was able to terminate radical chain reactions by converting free radicals to more stale products. Hence, SLTP showed more pronounced antioxidant activity than LTP. Degradation of TP into LTP could also improve the hydrogen atom donation effect. This might be the reason that LTP showed better antioxidant activity than TP.

Conclusions
The scavenging activity of superoxide anion radical, DPPH radical and hydroxyl radical were evaluated in this study. The results showed that degradation of TP could improve its water solubility and radical scavenging activity. Sulfation of LTP results in increased antioxidant activity than the natural LTP and TP. The results strongly support the fact that modification of LTP by sulfation will augment the antioxidant activity. Therefore, LTP and SLTP can be taken as potential natural resource of antioxidant, While, the structure-activities of SLTP and the activities in vivo need to be further investigated.