Characterization and Antioxidant Activities of Polysaccharides from Schisandra Chinensis
Xiaoxv Gao, Jianguang Chen*
Beihua University, Jilin 132013 , P.R. China
*Corresponding author. Email:
Abstract:SCPS, the water-soluble crude polysacchar i de extracted from the fru i ts of Sch i sandra ch i nens i s, was puri fi ed by DEAE-cellulo and Sepharo CL-6B column, obtai ned three fracti ons named as SCPS-1a, SCPS-1b and SCPS-2. In th i s paper, the chem i cal and phys i cal characteri sti cs of polysacchari de fracti ons and anti oxi dant capaci ty were evaluated. Experi mental results showed that SCPS-2 exh i b i ted ant i ox i dant act i v i ty s i gn i f i cantly i n a concentration-dependent manner, which can be developed as new antioxidant agents.
Key word: Schisandra chinensis; Polysacchar i de; Purification; Characterization; Antioxidant activity.
I.I NTRODUCTION
Schisandra chinensis (Turcz.) Baill, distributed abundantly in China, Korea and Japan, is famous for its
fruits’delicious taste, is the commercially important fruit and medicine, which has been ud as traditional Chine medicine from ancient China. It is officially listed and indexed as a dative and tonic, and also listed in the ‘Shen Nong Ben Cao Jing’, as a superior drug that can treat asthma and cure in coughs. According to Chine traditional medical science, the drug has warm and sour properties.
Recently, more and more rearches on free radicals have confirmed that traditional Chine medicine (TCM) rich in antioxidants play an esntial role in the prevention of neurodegenerative dias, cancers, cardiovascular dias, inflammation and other free radical induced problems[1], and arch for new antioxidants from TCM has achieved considerable attention [2]. Polysaccharides are important natural products from TCM, which exhibit significant antioxidant activities [3]. In the last decades, much evidence shows that the polysaccharides of S. chinensis are important active component. The rearch results are important cues for further understanding the pharmacological basis of S. chinensis as an antiaging, anticancer, anti-inflammation, and tonic drug. However, there have been ldom reports on free radical scavenging activities of polysaccharide from S. chinensis.
With the purpo of developing and extending the potential u of S. chinensis in antioxidant medici
ne, the prent rearch was carried out to investigate antioxidant activities of polysaccharide fractions from S. chinensis, as well as their chemical and physical characteristics.
II.M ATERIALS AND METHODS
A.Materials and chemicals
Schisandra chinensis was purchad from a local medicine market.
DEAE-cellulo and 1,1-diphenyl-2-picrylhydrazyl (DPPH) were purchad from Sigma (St. Louis, MO, USA). Sepharo CL-6B was purchad from Amersham Pharmacia Co. (Sweden). All other chemical reagents ud were analytical grade.
B.Purification of polysaccharide fractions
The fruits of Schisandra chinensis were cleaned and extracted with distilled water. The whole extract was filtered, centrifuged, and then precipitated with ethanol. The polysaccharide precipitate was collected and deproteinated by a combination of proteina and Sevag method [4], and then obtained crude Schisandra chinensis polysaccharides (cSCPS).
The cSCPS was dissolved, then loaded to DEAE–cellulo column, eluted successively with distilled water and 0.5 M NaCl. Fractions were monitored with the phenol–sulfuric acid method. The two main fractions (SCPS-1 and SCPS-2) was collected, dialyzed, and were further loaded on a Sepharo CL-6B column, eluted with 0.15 M NaCl to yield three main fractions, codes as SCPS-1a, SCPS-1b and SCPS-2. Fractions were dialyzed and lyophilized.
C.Molecular weight of polysaccharide fractions
Molecular weights were determined by HPLC. The polysaccharide fractions were loaded to Agilent HPLC system (Agilent Technologies, USA) with a TSK-G EL G3000 column, eluted with 0.1 mol/L Na2SO4 and detected by a RID-10A RID.
D.Monosaccharide compositions analysis
Polysaccharide were acetylated according to Lehrfeld [5]. Simply, the samples were hydrolyzed, and then reduced with KBH4, followed by neutralization. After adding myo-inositol and Na2CO3, then added pyridine–propylamine, and acetylated. Then, the acetylated products were analyzed using gas chromatography (GC).
E.Analysis of protein and carbohydrate contents
Total carbohydrate was analyzed by phenol-sulfuric acid colorimetric method [6]. Protein contents were quantified using Bradford’s method [7]. Total uronic acid content was analyzed by m-hydroxydiphenyl method [8]. F.DPPH free radical scavenging activity
Radical scavenging activity against DPPH was measured by the method [9] with a minor modification. Samples were dissolved in distilled water at the concentrations of 0, 0.5, 1, 2, 4, and 8 mg/mL. Samples (1ml) were mixed with 2 mL DPPH (0.1 mM, freshly prepared), then incubated at 25 for 30 min in the dark. The absorbance was measured at 517 nm. The scavenging activity was calculated as following formula:
2011 International Conference on Human Health and Biomedical Engineering August 19-22, 2011, Jilin, China
Scavenging effect (%) =(1 - Asample/Acontrol) ×
100%.dareto
G.Superoxide radical scavenging assay
The superoxide radicals scavenging effects of polysaccharide fractions were assayed by photoreduc
tion of NBT method [10]. 60ȝM phenazine methosulfate (PMS), 468ȝM nicotinamide adenine dinucleotide (NADH), 150 mM nitroblue tetrazolium (NBT), and various concentrations of samples were mixed in a volume of 3 ml. The mixture reacted at 20 for 10 min and then the absorbance was measured at 560 nm. The scavenging capability was calculated by the following equation: Scavenging effect (%) = (1íAsample/Acontrol) ×100%.
III.R ESULTS AND DISCUSSION
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A.Isolation and purification of polysaccharides
The yield of the crude polysaccharide extracted from Schisandra chinensis was 9.1 %. After deproteinated, the crude polysaccharide sample (cSCPS) was purified by DEAE-cellulo column (Fig.1), de-ionized water was ud to elute the unbound component (SCPS-1); and the retained components (SCPS-2) was eluted with 0.5 M NaCl. Then SCPS-1 and SCPS-2 were purified by Sephacryl S-300 column. Three main fractions (SCPS-1a, SCPS-1b and SCPS-2) were parated for further analysis. B.Physicochemical properties and chemical
compositions
The protein, total sugar, uronic acid contents, monosaccharides composition and molecular weight of all the fractions were summarized in Table 1. The total carbohydrate contents of SCPS-1a, SCPS-1b and SCPS-2 were 95.6%, 94.3% and 95.1%, respectively. According to Bradford method, the protein contents of SCPS-1b were 1.7%, not detected in SCPS-1a and SCPS-2. The results showed that the uronic acid contents in SCPS-2 were 28.7%, not detected in SCPS-1a and SCPS-1b. The average molecular weights of SCPS-1a, SCPS-1b and SCPS-2 calculated by HPLC, were 33.5 kDa, 86,4 kDa and 42,5 kDa, respectively. Bad on GC analysis, SCPS-
1a and SCPS-1b were both compod of four monosaccharides: arabino, xylo, manno and gluco with molar ratios of 15:8:10:32 and 18:13:10:41, respectively. SCPS-2 was compod of arabino, xylo, manno, gluco and glucuronic acid, the molar ratios are 12:4:10:35:37.
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Fig. 1. Polysaccharide fractionations isolated from Schisandra chinensis with DEAE-cellulo
(a) and Sepharo CL-6B (b, c). Crude polysaccharide (cSCPS) was purified on the DEAE-
cellulo column, distilled water was applied to elute the component (SCPS-1); and the retained components (SCPS-2) was eluted with NaCl. Then SCPS-1 and SCPS-2 were applied on Sepharo CL-6B, respectively, eluted with 0.15 M NaCl, obtained three main fractions
(SCPS-1a, SCPS-1b and SCPS-2)
C.DPPH radical scavenging activity
Fig. 2 demonstrated DPPH scavenging activity caud by polysaccharide fractions at different concentrations. SCPS-1a, SCPS-1b and SCPS-2 exhibited significant radical scavenging activities. The scavenging activity incread steadily at the concentration rang of 0.5–8 mg/mL. The DPPH radical scavenging rate of SCPS-1a, SCPS-1b and SCPS-2 reached 61.5%, 66.3% and 60.9% at 8 mg/mL, respectively. There was no significant difference in scavenging activity between SCPS-1a, SCPS-1b and SCPS-2 at the concentration rang of 0.5–8 mg/mL (p >0.05). The concentration required to inhibit 50% radical scavenging effect (IC50) was determined from the results of a ries of concentrations mentioned above. A lower IC50
value corresponds to a greater scavenging activity. The IC 50 values of SCPS-1a, SCPS-1b and SCPS-2 were 3.8, 3.6, and 3.7 mg/ml, respectively.
TABLE I. COMPOSITION AND M OLECULAR WEIGHT OF POLYSACCHARIDE FRACTIONS ISOLATED FROM S CHISANDRA CHINENSIS SCPS-1a SCPS-1b SCPS-2
Molecular weight 33,500 86,400 42,500
Total sugar (%) 95.6 94.3 95.1
Protein (%) nd 1.7 nd
Uronic acid (%) nd a nd 28.7 Sugar components (mol%) Arabino 23 18 12 Xylo 15 13 4 Manno 10 10 10
G luco 32 41 35
Glucuronic acid nd nd 37
nd: not detect
D. Superoxide radical scavenging activity
As shown in Fig. 3, superoxide scavenging activities of three fractions incread significantly (P < 0.01) with increasing concentrations from 0.5 mg/mL to 8.0 mg/mL, and the superoxide radical scavenging rate of SCPS-1a, SCPS-1b and SCPS-2 at 8.0 mg/mL was 51.5%, 46.2% and 54.7%, respectively. The IC 50 values of SCPS-1a, SCPS-1b and SCPS-2 were 7.7, 8.6, and 7.4 mg/ml,
Fig. 2.against DPPH radical. Results were prented as means ±S.D. (n=3).
IV. C ONCLUSIONS
Highly reactive free radicals and reactive oxygen species (ROS) commonly exist in the biological systems of live organs, which can oxidize nucleic acids, proteins, lipids
Fig. 3. Scavenging rate of different polysaccharide fractions isolated from Schisandra chinensis
against superoxide radical. Results were prented as means ±S.D. (n=3).
Polysaccharides can maintain human healthy, prevent and
retard the progress of many free radical-induced chronic
dias. In this paper, three polysaccharide fractions
(SCPS-1a, SCPS-1b and SCPS-2)from the fruits of
Schisandra chinensis were obtained by DEAE-cellulo and Sepharo CL-6B chromatography. In vitro models were applied to evaluate the antioxidant potential of S. chinensis polysaccharides. Expe
riment results showed that all fractions exhibited significant antioxidant activity in a concentration-dependent manner..
A CKNOWLEDGMENT
This work was funded by Scientific Rearch Fund of
Education Office of Jilin Province (2011-128).
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