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Abstract [Objectives] This study was conducted to optimize the extraction process of Sipunculus nudus polysaccharide. [Methods] S. nudus polysaccharide was extracted by ultrasonic water extraction， with the marine S. nudus as a raw material from Sanya City， Hainan Province， by single factor variable method， to find the optimal extraction conditions including ultrasonic power， solid-liquid ratio， ultrasonic extraction temperature， ultrasonic extraction time， ultrasonic extraction times and lye concentration. The absorbance of the polysaccharide was determined by an ultraviolet spectrophotometer， and polysaccharide yield were calculated. The antioxidant activity of scavenging hydroxyl and DPPH radicals was measured at different concentrations. [Results] The optimal extraction conditions for S. nudus polysaccharide were as follows： solid-liquid ratio at 1∶11 mg/ml， extraction temperature at 60 ℃， three times of ultrasonic extraction， ultrasonic power of 960 W， extraction time of 1.5 h， and lye concentration of 5%， under which the polysaccharide yield was 3.21%. The scavenging rates of hydroxyl radical and DPPH radical were 12.58% at the concentration of 0.20 mg/ml. Moreover， the scavenging rates increased significantly with the increase of concentration. [Conclusions] This method is a new trial with satisfactory results.
Key words Sipunculus nudus； Ultrasound； Lye concentration； Polysaccharide； Antioxidant activity
Sipunculus nudus， commonly known as sandworm， is a kind of invertebrate in the shape of naked bowel without hair， the body wall of which has bundled longitudinal muscle[1]. It is widely distributed in the world， grows along coastal mud flat. Once its growing environment is polluted， it could not survive. S. nudus is very sensitive to environmental quality， and thus called as "the mark organism of environment"[2]. Polysaccharide is a kind of carbohydrate with complicated structure formed by condensation and dehydration of multiple monosaccharide molecules， and there are various kinds of polysaccharides， which are mainly distributed in animals and plants. Researches show that marine life polysaccharides are mainly divided into three kinds， i.e.， marine microbial polysaccharides， algal polysaccharides and marine animal polysaccharides[3-6]. The polysaccharide extraction methods are mainly divided into conventional methods and modern methods， among which the conventional methods mainly include hot water extraction and cold water extraction methods and acid-base solution method， which have the disadvantage of low efficiency and cause hydrolysis of polysaccharides easily. Modern methods include ultrasonic-assisted extraction， microwave-assisted method and enzymolysis method[7]. In this study， S. nudus polysaccharide was extracted by ultrasonic alkali extraction method， which destroys cells of extracted materials through special action such as strong vibration， high accelerated speed， cavitation effect and stirring and allows the penetration of solvent into cells and thus the dissolution of effective components in extracted cells. The method has the advantages of short extraction time and high efficiency[8]， and could the consumption of raw materials and reagents. Materials and Methods
Materials and instruments
Fresh and alive S. nudus was purchased from Sanya City.
Standard solution of glucose， anhydrous ethanol， 95% ethanol， sodium hydroxide， concentrated sulfuric acid， phenol， hydrochloric acid， acetone， trichloroacetic acid， diethyl ether， phenolphthalein， petroleum ether， trichloromethane， iodine bromide， potassium iodide， sodium thiosulfate， acetic anhydride， glacial acetic acid， potassium hydroxide， ferrous sulfate， hydrogen peroxide， salicylic acid， DPPH， starch indicator， unless otherwise indicated， were all analytical pure， and purchased from Tianjin Fuyu Fine Chemicals Co.， Ltd. The used water was ultrapure water.
JJ-2 triturating machine （Jintan Baita Xinbao Instrument Company）； electronic balance FA2204B （Techcomp Precision Balance Instrument Co.， Ltd.）； regulating type ultrasonic generator JU-6224 （Shanghai JUMP Ultrasonic Equipment Co.， Ltd.）； high-speed table centrifuge TGL-18C （Jiangsu Shenglan Instrument Manufacturing Co.， Ltd.）； vacuum dryer DZF-OB （Jiangsu Shenglan Instrument Manufacturing Co.， Ltd.）； thermostat magnetic stirrer X85-2S （Jiangsu Shenglan Instrument Manufacturing Co.， Ltd.）； HH-digital thermostat water bath （Jiangsu Shenglan Instrument Manufacturing Co.， Ltd.）； UV-2550 ultraviolet spectrophotometer （Shimadzu， Japan）.
Methods
Pretreatment of S. nudus
Fresh and alive S. nudus were cut open to remove viscera and cleaned. It was cleaned with distilled water for 2-3 times， cut into pieces， and triturated in a JJ-2 triturating machine.
Use of S. nudus raw material
The triturated S. nudus was stored in a refrigerator for later use. The material was put into the refrigerator immediately after use to avoid deterioration.
Determination of polysaccharides from S. nudus
Wavelength for spectrum scanning of glucose
The standard curve of glucose was determined by sulfuric acid-phenol method. A certain amount of analytically pure glucose was added into a drying oven and dried at 100 ℃ to constant weight. Then， 0.267 6 g of the glucose was added into distilled water to dissolve it， and the solution was then transferred to 100 ml of volumetric flask， and shaken uniformly. From the obtained solution， 5 ml was added into a 25 ml colorimetric tube and added with distilled water to constant volume. Into 10 ml of dry test tubes， 0， 0.2， 0.4， 0.6， 0.8 and 1.0 ml were added， respectively， followed by the addition of pure water to 1 ml. Then， 2.0 ml of 5% phenol and 3.0 ml concentrated sulfuric acid were added， followed by shaking and cooling to room temperature. The solutions were then scanned with a TU-1901 double-beam ultraviolet and visible spectrophotometer. Plotting of glucose standard curve
Standard solutions of glucose with different concentrations were prepared and determined under specific wavelength， and a standard curve was plotted with concentrations as horizontal coordinates and absorbance as vertical coordinates. The crude polysaccharide extracted by ultrasonic water extraction was dissolved by the same method and determined for absorbance， and the polysaccharide concentration was then obtained according to the standard curve.
Extraction of S. nudus polysaccharide by ultrasonic water extraction
Extraction process of S. nudus polysaccharide： Ultrasonic water extraction was performed for various experiments， and the extract was centrifuged at 10 000 r/min for 30 min， and the supernatants were added with trichloroacetic acid to remove protein， and regulated to a pH value of 4. After standing for about 10 min， the supernatants were centrifuged at 4 000 r/min， and the obtained supernatants were concentrated at 90 ℃ in a thermostatic water bath to about 1/4 of the original volumes[12]. Then， 95% ethanol was added according to a ratio of 1∶3 （concentrate：95% ethanol）， and after standing at 4 ℃ for 12 h， the precipitate was filtered out， cleaned with anhydrous ethanol and acetone sequentially， and naturally air-dried， obtaining crude polysaccharide. The crude polysaccharide obtained by ultrasonic water extraction was prepared according to the preparation method of the standard solutions of glucose into solutions， which were diluted and determined for absorbance. According to the standard curve， the concentration of S. nudus crude polysaccharide could be obtained， and the yield of S. nudus polysaccharide could be then obtained by calculation.
Polysaccharide yield （%）=CxV1xV2x100[9]MxV0x1000
Wherein C is determined polysaccharide concentration； V1 is the volume of crude polysaccharide solution； V2 is the volume after dilution； V0 is the volume for determination； and M is the mass of S. nudus.
Effect of ultrasonic power on extraction of S. nudus polysaccharide
According to a solid-liquid ratio of 1∶8 mg/ml， the homogenate of S. nudus was accurately added into a 100 ml conical flask， and added with distilled water. Ultrasonic extraction was performed twice at 50 ℃ for 1 h （1 h/time）， under the ultrasonic power conditions of 750， 900， 1 050， 1 200 and 1 350 W， respectively， to investigate the effect of ultrasonic power on the extraction rate of S. nudus polysaccharide， so as to determine the optimal ultrasonic power. Effect of solid-liquid ratio on extraction of S. nudus polysaccharide
The homogenate of S. nudus was accurately added into a 100 ml conical flask， and under the temperature of 70 ℃， ultrasonic power of 750 W， ultrasonic extraction time of 1 h， and two times of extraction （1 h/time）， the effects of different solid-liquid ratios of 1∶5， 1∶8， 1∶11， 1∶14， 1∶17 and 1∶20 mg/ml on the extraction rate of S. nudus polysaccharide were investigated， so as to determine the optimal solid-liquid ratio.
Effect of ultrasonic extraction temperature on extraction of S. nudus polysaccharide
According to the solid-liquid ratio of 1∶8 mg/ml， the homogenate of S. nudus was accurately added into a 100 ml conical flask， and added with distilled water. Under the ultrasonic power of 750 W， ultrasonic extraction time of 1 h， and two times of ultrasonic extraction （1 h/time）， extraction was performed at 40， 50， 60， 70， and 80 ℃， respectively， to investigate the effects of different extraction temperatures on the extraction rate of polysaccharide， so as to determine the optimal ultrasonic extraction temperature.
Effect of ultrasonic extraction time on extraction of S. nudus polysaccharide
According to the solid-liquid ratio of 1∶8 mg/ml， the homogenate of S. nudus was accurately added into a 100 ml conical flask， and added with distilled water. Under the ultrasonic power of 750 W， ultrasonic extraction temperature of 60 ℃ and two times of ultrasonic extraction， ultrasonic extraction was performed for 0.5， 1.0， 1.5， 2.0， 2.5 and 3.0 h， respectively， to investigate the effects of different extraction time on the extraction rate of polysaccharide， so as to determine the optimal ultrasonic extraction time.
Effect of ultrasonic extraction times on extraction of S. nudus polysaccharide
According to the solid-liquid ratio of 1∶8 mg/ml， the homogenate of S. nudus was accurately added into a 100 ml conical flask， and added with distilled water. Under the ultrasonic power of 750 W， ultrasonic extraction temperature of 60 ℃ and extraction time of 1 h， the sample was extracted for one， two， three and four times， respectively， to investigate the effects of different extraction times on the extraction rate of polysaccharide， so as to determine the optimal ultrasonic extraction times.
election of lye concentration
Lye concentrations were set as 4%， 5%， 6%， 7% and 8%. According to a solid-liquid ratio of 1∶6 g/ml， materials were accurately weighed and subjected to polysaccharide extraction under 960 W at 60 ℃ for 2 h. The optimal lye concentration was determined according to polysaccharide yield. Results and Analysis
Plotting of standard curve of glucose
Determination of wavelength for scanning of standard solutions of glucose
As shown in Fig. 1， standard solutions of glucose were scanned by above experimental method， and a better spectral band was selected as the absorption wavelength. According to the obtained spectra， 491 nm was selected as the wavelength for the plotting of standard curve of glucose.
Standard curve of glucose solutions
As shown in Fig. 2， the absorbance values of the various standard solutions of glucose were determined by above experimental method， and the standard curve was plotted by computer， obtaining the regression equation of the standard curve of glucose and the regression coefficient. The regression equation of the standard curve of glucose was y=2.743x-0.012 53 （R2=0.999 4）， indicating good precision of the curve.
Effect of ultrasonic power on the extraction rate of S. nudus polysaccharide
As shown in Fig. 3， the crude polysaccharide products of the various experiment groups were dissolved with distilled water， and transferred to 10 ml test tubes and diluted to constant volume， respectively. From each of the test tube， 1 ml was taken and diluted to 5 ml， 1/5 of which， i.e.， 1 ml was taken for later use. The absorbance values of the solutions were determined according to the method of the standard solutions of glucose， and polysaccharide yields were calculated. When other extraction conditions were fixed， with the ultrasonic power increasing， the extraction rate of S. nudus polysaccharide increased and reached its maximum value at 960 W， beyond which the extraction rate of S. nudus polysaccharide decreased gradually with the continuous increase of ultrasonic power. It was determined during the single factor investigation of ultrasonic power that the optimal ultrasonic power was 960 W.
Effect of solid-liquid ratio on the extraction rate of S. nudus polysaccharide
As shown in Fig. 4， the crude polysaccharide products of the various experiment groups were dissolved with distilled water， and transferred to 10 ml test tubes and diluted to constant volume， respectively. From each of the test tube， 1 ml was taken and diluted to 5 ml， 1/5 of which， i.e.， 1 ml was taken for later use. The absorbance values of the solutions were determined according to the method of the standard solutions of glucose， and polysaccharide yields were calculated. With the solid-liquid ratio increasing， the polysaccharide yield reached the highest value of 0.86% at the solid-liquid ratio of 1∶11 mg/ml， and then， with the continuous increase of the solid-liquid ratio， the polysaccharide yield decreased， then increased and decreased again. Such results are due to that too-little solvent could not dissolve all the S. nudus， i.e.， soluble polysaccharide could not thoroughly run out， but when the solvent is too much， the concentration is too low， which affects polysaccharide yield. Therefore， the optimal solid-liquid ratio was determined to be 1∶11 mg/ml. Effect of ultrasonic extraction temperature on the extraction rate of S. nudus polysaccharide
As shown in Fig. 5， the crude polysaccharide products of the various experiment groups were dissolved with distilled water， and transferred to 10 ml test tubes and diluted to constant volume， respectively. From each of the test tube， 1 ml was taken and diluted to 5 ml， 1/5 of which， i.e.， 1 ml was taken for later use. The absorbance values of the solutions were determined according to the method of the standard solutions of glucose， and polysaccharide yields were calculated. With the increase of temperature， the polysaccharide also increased， and reached its maximum value of 0.38% at 60 ℃， beyond which the extraction rate decreased with the continuous increase of temperature. Such results are due to that for ultrasonic treatment， the temperature should not be too high， and a too-high temperature would cause the degradation of polysaccharide[10-12]. The optimal extraction temperature was determined to be 60 ℃.
Effect of ultrasonic extraction time on the extraction rate of S. nudus polysaccharide
As shown in Fig. 6， the crude polysaccharide products of the various experiment groups were dissolved with distilled water， and transferred to 10 ml test tubes and diluted to constant volume， respectively. From each of the test tube， 1 ml was taken and diluted to 5 ml， 1/5 of which， i.e.， 1 ml was taken for later use. The absorbance values of the solutions were determined according to the method of the standard solutions of glucose， and polysaccharide yields were calculated. At first， the polysaccharide yield increased over time， and reached its maximum value of 0.31% at 1.5 h， and afterwards， the value decreased over time instead. Such results are due to that ultrasound has stronger mechanical cutting action， and long time action would destruct polysaccharide， causing loss of polysaccharide， which further affects polysaccharide yield[13-17]. The optimal ultrasonic extraction time was determined as 1.5 h.
Effect of ultrasonic extraction times on the extraction rate of S. nudus polysaccharide
As shown in Fig. 7， the crude polysaccharide products of the various experiment groups were dissolved with distilled water， and transferred to 10 ml test tubes and diluted to constant volume， respectively. From each of the test tube， 1 ml was taken and diluted to 5 ml， 1/5 of which， i.e.， 1 ml was taken for later use. The absorbance values of the solutions were determined according to the method of the standard solutions of glucose， and polysaccharide yields were calculated. At first， with the increase of ultrasonic extraction times， the polysaccharide yield increased and reached its maximum value of 1.19% under three times of extraction， and then， the yield decreased with the extraction times increasing instead. Such results are due to that ultrasound has the effect of breaking cells by shaking， and after extracting more than three times， other substances effuse， which affects the yield of polysaccharide. Effect of lye concentration on the extraction rate of S. nudus polysaccharide
As shown in Fig. 8， the crude polysaccharide products of the various experiment groups were prepared and determined according to the experimental operations for polysaccharide solution preparation and determination， and polysaccharide yields were calculated. At first， with the lye concentration increasing， the polysaccharide yield increased and reached its maximum value at the lye concentration of 5%， beyond which the yield decreased with the continuous increase of lye concentration. The results are due to that a too-high lye concentration would degrade polysaccharide， which further reduces the extraction rate. It could be seen that the optimal lye concentration was 5%.
Univariate factors were investigated by single-factor variable method， and the optimal extraction conditions for S. nudus polysaccharide were as follows： solid-liquid ratio at 1∶11 mg/ml， extraction temperature at 60 ℃， three times of ultrasonic extraction， ultrasonic power of 960 W， extraction time of 1.5 h， and lye concentration of 5%， under which the polysaccharide yield was 3.21%.
Determination of antioxidant activity of S. nudus polysaccharide
S. nudus polysaccharide activity of scavenging hydroxyl radical
Determination of hydroxyl radical scavenging capacity of S. nudus polysaccharide
A proper amount of S. nudus polysaccharide was transferred into a dry 100.00 ml beaker， and 0.120 0 g of S. nudus polysaccharide was accurately weighed with an electronic analytical balance. Into the beaker， 10.00 ml of anhydrous ethanol was added， to dissolve all the S. nudus polysaccharide. The solution was transferred into a 100.00 ml volumetric flask， the beaker was flushed with 10.00 ml of anhydrous ethanol for at least 5-6 times， and the flushing fluid was also poured into the 100.00 ml volumetric flask. Then， anhydrous ethanol was added into the volumetric flask to constant volume， obtaining 1.20 mg/ml S. nudus polysaccharide ethanol solution. Next， the 1.20 mg/ml S. nudus polysaccharide ethanol solution was diluted sequentially， obtaining 0.20， 0.40， 0.60， 0.80 and 1.00 mg/ml S. nudus polysaccharide ethanol solutions. Into 6 marked 100 ml conical flasks， 2.00 ml of the different concentrations of S. nudus polysaccharide ethanol solutions were added， respectively， and next， 1.00 ml of 0.010 0 mol/L FeSO4 solution and 1.50 ml of 10% hydrogen peroxide were then added sequentially. After continuous shaking and 10 min of standing， 2.00 ml of 0.0100 mol/L salicylic acid was added to the 6 conical flasks sequentially， and the solutions were stood for 30 min for later use. Each group of the testing samples were scanned with an ultraviolet spectrophotometer， and the optimal wavelength was 510 nm for all of them. Then， at the wavelength of 510 nm， different concentrations of S. nudus polysaccharide ethanol solutions were determined for absorbance A1. According to the same method， into 6 marked 100 ml conical flasks， 2.00 ml of the different concentrations of S. nudus polysaccharide ethanol solutions were added， respectively， and next， 1.00 ml of 0.010 0 mol/L FeSO4 solution and 1.50 ml of distilled water were added sequentially. After continuous shaking and 10 min of standing， 2.00 ml of 0.010 0 mol/L salicylic acid was added to the 6 conical flasks sequentially， and the solutions were stood for 30 min for later use. The solutions were determined with the ultraviolet spectrometer at 510 nm sequentially， for absorbance A2. Also， into the third group of 6 marked 100 ml conical flasks， 2.00 ml of anhydrous ethanol was added instead of S. nudus polysaccharide ethanol solutions， respectively， and next， 1.00 ml of 0.010 0 mol/L FeSO4 solution and 1.50 ml of distilled water were added sequentially. After continuous shaking and 10 min of standing， 2.00 ml of 0.010 0 mol/L salicylic acid was added to the 6 conical flasks sequentially， and the solutions were stood for 30 min for later use. The solutions were determined at 510 nm by the same determination method， for absorbance A0. Meanwhile， with TBHQ as positive control， check test was carried out according to above steps， and the determined results were compared with the results of S. nudus polysaccharide ethanol solutions. Each group of test was designed with three replicates， and the experimental data were recorded and averaged. The hydroxyl radical scavenging rate of S. nudus polysaccharide was calculated according to the computational formula： Hydroxyl radical scavenging rate （%）=[A0- （A1-A2）]/A0×100[18-22]. The determined absorbance data of different concentrations of S. nudus polysaccharide to hydroxyl radical under different conditions were determined， as shown in Table 1. The hydroxyl radical scavenging rates of different concentrations of S. nudus polysaccharide and TBHQ were determined. It could be seen from Fig. 9 that the hydroxyl radical scavenging rate of S. nudus polysaccharide was remarkably lower than common antioxidant TBHQ， and increased with the increase of concentration， basically exhibiting linear relation. At the concentration of 0.20 mg/ml， the hydroxyl radical scavenging rates of S. nudus polysaccharide and TBHQ reached 12.58% and 31.09%， respectively， and hydroxyl radical scavenging rates of both of them increased with the increase of concentration. The regression equation of S. nudus polysaccharide was Y=24.290X+12.915 （R2=0.863 0）， and the regression equation of TBHQ was Y=48.826X+31.939 （R2=0.850 7）. The hydroxyl radical scavenging capacity of S. nudus polysaccharide was about 41%-50% of that of TBHQ， indicating that S. nudus polysaccharide also had very strong hydroxyl radical scavenging capacity， which is relatively lower than common antioxidant TBHQ.
DPPH radical scavenging activity of S. nudus polysaccharide
Determination of DPPH radical scavenging capacity of S. nudus polysaccharide
A proper amount of S. nudus polysaccharide was transferred into a dry 100.00 ml beaker， and 0.120 0 g of S. nudus polysaccharide was accurately weighed with an electronic analytical balance. Into the beaker， 10.00 ml of anhydrous ethanol was added， to dissolve all the S. nudus polysaccharide. The solution was transferred into a 100.00 ml volumetric flask， the beaker was flushed with 10.00 ml of anhydrous ethanol for at least 5-6 times， and the flushing fluid was also poured into the 100.00 ml volumetric flask. Then， anhydrous ethanol was added into the volumetric flask to constant volume， obtaining 1.20 mg/ml S. nudus polysaccharide ethanol solution. Next， the 1.20 mg/ml S. nudus polysaccharide ethanol solution was diluted sequentially， obtaining 0.20， 0.40， 0.60， 0.80 and 1.00 mg/ml S. nudus polysaccharide ethanol solutions. Into 6 marked 100 ml conical flasks， 2.00 ml of the different concentrations of S. nudus polysaccharide ethanol solutions were added， respectively， and next， 2.00 ml of 0.017 8 mol/L DPPH anhydrous ethanol solution was then added sequentially. The solutions were heated in water bath at 40 ℃ for 30 min， for later use. Each group of the testing samples were scanned with an ultraviolet spectrophotometer， and the optimal wavelength was 517 nm for all of them. Then， at the wavelength of 517 nm， different concentrations of S. nudus polysaccharide ethanol solutions were determined for absorbance A1. According to the same method， into other 6 marked 100 ml conical flasks， 2.00 ml of the different concentrations of S. nudus polysaccharide ethanol solutions were added， respectively， and next， 2.00 ml of anhydrous ethanol was then added sequentially. The solutions were heated in water bath at 40 ℃ for 30 min， and determined with the ultraviolet spectrometer at 517 nm for absorbance A2. Also， into the third group of 6 marked 100 ml conical flasks， 2.00 ml of anhydrous ethanol was added instead of S. nudus polysaccharide ethanol solutions， respectively， and next， 2.00 ml of 0.017 8 mol/L DPPH anhydrous ethanol solution was then added sequentially. The solutions were heated in water bath at 40 ℃ for 30 min， and determined with the ultraviolet spectrometer at 517 nm for absorbance A0. Meanwhile， with TBHQ as positive control， check test was carried out according to above steps， and the determined results were compared with the results of S. nudus polysaccharide ethanol solutions. Each group of test was designed with three replicates， and the experimental data were recorded and averaged. The DPPH radical scavenging rate of S. nudus polysaccharide was calculated according to the computational formula： DPPH radical scavenging rate （%）=[A0- （A1-A2）]/A0×100[18-22]. The determined absorbance data of different concentrations of S. nudus polysaccharide to DPPH radical under different conditions were determined， as shown in Table 2. DPPH radical scavenging activity of S. nudus polysaccharide
The DPPH scavenging rates of different concentrations of S. nudus polysaccharide and TBHQ were determined. It could be seen from Fig. 10 that the DPPH scavenging rate of S. nudus polysaccharide was remarkably lower than common antioxidant TBHQ， and increased with the increase of concentration， basically exhibiting linear relation. At the concentration of 0.20 mg/ml， the DPPH scavenging rates of S. nudus polysaccharide and TBHQ reached 12.58% and 32.78%， respectively， and DPPH scavenging rates of both of them increased with the increase of concentration. The regression equation of S. nudus polysaccharide was Y=28.551X+8.080 7 （R2=0.934 7）， and the regression equation of TBHQ was Y=61.633X+26.089 （R2=0.943 1）. The DPPH scavenging capacity of S. nudus polysaccharide was about 37%-44% of that of TBHQ， indicating that S. nudus polysaccharide also had very strong DPPH scavenging capacity， which is relatively lower than common antioxidant TBHQ.
Conclusions
Univariate factors were investigated by single factor variable method， and the optimal extraction conditions for S. nudus polysaccharide were as follows： solid-liquid ratio at 1∶11 mg/ml， extraction temperature at 60 ℃， three times of ultrasonic extraction， ultrasonic power of 960 W， extraction time of 1.5 h， and lye concentration of 5%， under which the polysaccharide yield was 3.21%. The scavenging activity of S. nudus polysaccharide against hydroxyl radical and DPPH radical under different concentrations was determined. At the concentration of 0.20 mg/ml， scavenging rates of S. nudus polysaccharide against hydroxyl radical and DPPH radical were both 12.58%， and the scavenging rates of both the two kinds of radicals increased with the increase of concentration. Through the comparison with the antioxidant activity of antioxidant TBHQ， the antioxidant scavenging capacity of S. nudus polysaccharide was studied in vitro. The hydroxyl radical and DPPH radical scavenging capacities of S. nudus polysaccharide exhibited a remarkable dose-effect relationship， and compared with antioxidant TBHQ at the same concentration， its scavenging capacities of above two kinds of radicals were lower than TBHQ， equivalent to 40%-50% of the effects of TBHQ， indicating that S. nudus polysaccharide has antioxidant activity， with very good effect， and it is directly demonstrated that the radical scavenging capacity of S. nudus polysaccharide is related to the contents of antioxidant components contained in it. S. nudus polysaccharide contains rich aromatics， unsaturated fatty acids， aldehydes， esters and alkenes， all of which have very strong reducibility and might be related to radical scavenging activity. The mechanism of the main functional components with radical scavenging activity still needs further study.
Key words Sipunculus nudus； Ultrasound； Lye concentration； Polysaccharide； Antioxidant activity
Sipunculus nudus， commonly known as sandworm， is a kind of invertebrate in the shape of naked bowel without hair， the body wall of which has bundled longitudinal muscle[1]. It is widely distributed in the world， grows along coastal mud flat. Once its growing environment is polluted， it could not survive. S. nudus is very sensitive to environmental quality， and thus called as "the mark organism of environment"[2]. Polysaccharide is a kind of carbohydrate with complicated structure formed by condensation and dehydration of multiple monosaccharide molecules， and there are various kinds of polysaccharides， which are mainly distributed in animals and plants. Researches show that marine life polysaccharides are mainly divided into three kinds， i.e.， marine microbial polysaccharides， algal polysaccharides and marine animal polysaccharides[3-6]. The polysaccharide extraction methods are mainly divided into conventional methods and modern methods， among which the conventional methods mainly include hot water extraction and cold water extraction methods and acid-base solution method， which have the disadvantage of low efficiency and cause hydrolysis of polysaccharides easily. Modern methods include ultrasonic-assisted extraction， microwave-assisted method and enzymolysis method[7]. In this study， S. nudus polysaccharide was extracted by ultrasonic alkali extraction method， which destroys cells of extracted materials through special action such as strong vibration， high accelerated speed， cavitation effect and stirring and allows the penetration of solvent into cells and thus the dissolution of effective components in extracted cells. The method has the advantages of short extraction time and high efficiency[8]， and could the consumption of raw materials and reagents. Materials and Methods
Materials and instruments
Fresh and alive S. nudus was purchased from Sanya City.
Standard solution of glucose， anhydrous ethanol， 95% ethanol， sodium hydroxide， concentrated sulfuric acid， phenol， hydrochloric acid， acetone， trichloroacetic acid， diethyl ether， phenolphthalein， petroleum ether， trichloromethane， iodine bromide， potassium iodide， sodium thiosulfate， acetic anhydride， glacial acetic acid， potassium hydroxide， ferrous sulfate， hydrogen peroxide， salicylic acid， DPPH， starch indicator， unless otherwise indicated， were all analytical pure， and purchased from Tianjin Fuyu Fine Chemicals Co.， Ltd. The used water was ultrapure water.
JJ-2 triturating machine （Jintan Baita Xinbao Instrument Company）； electronic balance FA2204B （Techcomp Precision Balance Instrument Co.， Ltd.）； regulating type ultrasonic generator JU-6224 （Shanghai JUMP Ultrasonic Equipment Co.， Ltd.）； high-speed table centrifuge TGL-18C （Jiangsu Shenglan Instrument Manufacturing Co.， Ltd.）； vacuum dryer DZF-OB （Jiangsu Shenglan Instrument Manufacturing Co.， Ltd.）； thermostat magnetic stirrer X85-2S （Jiangsu Shenglan Instrument Manufacturing Co.， Ltd.）； HH-digital thermostat water bath （Jiangsu Shenglan Instrument Manufacturing Co.， Ltd.）； UV-2550 ultraviolet spectrophotometer （Shimadzu， Japan）.
Methods
Pretreatment of S. nudus
Fresh and alive S. nudus were cut open to remove viscera and cleaned. It was cleaned with distilled water for 2-3 times， cut into pieces， and triturated in a JJ-2 triturating machine.
Use of S. nudus raw material
The triturated S. nudus was stored in a refrigerator for later use. The material was put into the refrigerator immediately after use to avoid deterioration.
Determination of polysaccharides from S. nudus
Wavelength for spectrum scanning of glucose
The standard curve of glucose was determined by sulfuric acid-phenol method. A certain amount of analytically pure glucose was added into a drying oven and dried at 100 ℃ to constant weight. Then， 0.267 6 g of the glucose was added into distilled water to dissolve it， and the solution was then transferred to 100 ml of volumetric flask， and shaken uniformly. From the obtained solution， 5 ml was added into a 25 ml colorimetric tube and added with distilled water to constant volume. Into 10 ml of dry test tubes， 0， 0.2， 0.4， 0.6， 0.8 and 1.0 ml were added， respectively， followed by the addition of pure water to 1 ml. Then， 2.0 ml of 5% phenol and 3.0 ml concentrated sulfuric acid were added， followed by shaking and cooling to room temperature. The solutions were then scanned with a TU-1901 double-beam ultraviolet and visible spectrophotometer. Plotting of glucose standard curve
Standard solutions of glucose with different concentrations were prepared and determined under specific wavelength， and a standard curve was plotted with concentrations as horizontal coordinates and absorbance as vertical coordinates. The crude polysaccharide extracted by ultrasonic water extraction was dissolved by the same method and determined for absorbance， and the polysaccharide concentration was then obtained according to the standard curve.
Extraction of S. nudus polysaccharide by ultrasonic water extraction
Extraction process of S. nudus polysaccharide： Ultrasonic water extraction was performed for various experiments， and the extract was centrifuged at 10 000 r/min for 30 min， and the supernatants were added with trichloroacetic acid to remove protein， and regulated to a pH value of 4. After standing for about 10 min， the supernatants were centrifuged at 4 000 r/min， and the obtained supernatants were concentrated at 90 ℃ in a thermostatic water bath to about 1/4 of the original volumes[12]. Then， 95% ethanol was added according to a ratio of 1∶3 （concentrate：95% ethanol）， and after standing at 4 ℃ for 12 h， the precipitate was filtered out， cleaned with anhydrous ethanol and acetone sequentially， and naturally air-dried， obtaining crude polysaccharide. The crude polysaccharide obtained by ultrasonic water extraction was prepared according to the preparation method of the standard solutions of glucose into solutions， which were diluted and determined for absorbance. According to the standard curve， the concentration of S. nudus crude polysaccharide could be obtained， and the yield of S. nudus polysaccharide could be then obtained by calculation.
Polysaccharide yield （%）=CxV1xV2x100[9]MxV0x1000
Wherein C is determined polysaccharide concentration； V1 is the volume of crude polysaccharide solution； V2 is the volume after dilution； V0 is the volume for determination； and M is the mass of S. nudus.
Effect of ultrasonic power on extraction of S. nudus polysaccharide
According to a solid-liquid ratio of 1∶8 mg/ml， the homogenate of S. nudus was accurately added into a 100 ml conical flask， and added with distilled water. Ultrasonic extraction was performed twice at 50 ℃ for 1 h （1 h/time）， under the ultrasonic power conditions of 750， 900， 1 050， 1 200 and 1 350 W， respectively， to investigate the effect of ultrasonic power on the extraction rate of S. nudus polysaccharide， so as to determine the optimal ultrasonic power. Effect of solid-liquid ratio on extraction of S. nudus polysaccharide
The homogenate of S. nudus was accurately added into a 100 ml conical flask， and under the temperature of 70 ℃， ultrasonic power of 750 W， ultrasonic extraction time of 1 h， and two times of extraction （1 h/time）， the effects of different solid-liquid ratios of 1∶5， 1∶8， 1∶11， 1∶14， 1∶17 and 1∶20 mg/ml on the extraction rate of S. nudus polysaccharide were investigated， so as to determine the optimal solid-liquid ratio.
Effect of ultrasonic extraction temperature on extraction of S. nudus polysaccharide
According to the solid-liquid ratio of 1∶8 mg/ml， the homogenate of S. nudus was accurately added into a 100 ml conical flask， and added with distilled water. Under the ultrasonic power of 750 W， ultrasonic extraction time of 1 h， and two times of ultrasonic extraction （1 h/time）， extraction was performed at 40， 50， 60， 70， and 80 ℃， respectively， to investigate the effects of different extraction temperatures on the extraction rate of polysaccharide， so as to determine the optimal ultrasonic extraction temperature.
Effect of ultrasonic extraction time on extraction of S. nudus polysaccharide
According to the solid-liquid ratio of 1∶8 mg/ml， the homogenate of S. nudus was accurately added into a 100 ml conical flask， and added with distilled water. Under the ultrasonic power of 750 W， ultrasonic extraction temperature of 60 ℃ and two times of ultrasonic extraction， ultrasonic extraction was performed for 0.5， 1.0， 1.5， 2.0， 2.5 and 3.0 h， respectively， to investigate the effects of different extraction time on the extraction rate of polysaccharide， so as to determine the optimal ultrasonic extraction time.
Effect of ultrasonic extraction times on extraction of S. nudus polysaccharide
According to the solid-liquid ratio of 1∶8 mg/ml， the homogenate of S. nudus was accurately added into a 100 ml conical flask， and added with distilled water. Under the ultrasonic power of 750 W， ultrasonic extraction temperature of 60 ℃ and extraction time of 1 h， the sample was extracted for one， two， three and four times， respectively， to investigate the effects of different extraction times on the extraction rate of polysaccharide， so as to determine the optimal ultrasonic extraction times.
election of lye concentration
Lye concentrations were set as 4%， 5%， 6%， 7% and 8%. According to a solid-liquid ratio of 1∶6 g/ml， materials were accurately weighed and subjected to polysaccharide extraction under 960 W at 60 ℃ for 2 h. The optimal lye concentration was determined according to polysaccharide yield. Results and Analysis
Plotting of standard curve of glucose
Determination of wavelength for scanning of standard solutions of glucose
As shown in Fig. 1， standard solutions of glucose were scanned by above experimental method， and a better spectral band was selected as the absorption wavelength. According to the obtained spectra， 491 nm was selected as the wavelength for the plotting of standard curve of glucose.
Standard curve of glucose solutions
As shown in Fig. 2， the absorbance values of the various standard solutions of glucose were determined by above experimental method， and the standard curve was plotted by computer， obtaining the regression equation of the standard curve of glucose and the regression coefficient. The regression equation of the standard curve of glucose was y=2.743x-0.012 53 （R2=0.999 4）， indicating good precision of the curve.
Effect of ultrasonic power on the extraction rate of S. nudus polysaccharide
As shown in Fig. 3， the crude polysaccharide products of the various experiment groups were dissolved with distilled water， and transferred to 10 ml test tubes and diluted to constant volume， respectively. From each of the test tube， 1 ml was taken and diluted to 5 ml， 1/5 of which， i.e.， 1 ml was taken for later use. The absorbance values of the solutions were determined according to the method of the standard solutions of glucose， and polysaccharide yields were calculated. When other extraction conditions were fixed， with the ultrasonic power increasing， the extraction rate of S. nudus polysaccharide increased and reached its maximum value at 960 W， beyond which the extraction rate of S. nudus polysaccharide decreased gradually with the continuous increase of ultrasonic power. It was determined during the single factor investigation of ultrasonic power that the optimal ultrasonic power was 960 W.
Effect of solid-liquid ratio on the extraction rate of S. nudus polysaccharide
As shown in Fig. 4， the crude polysaccharide products of the various experiment groups were dissolved with distilled water， and transferred to 10 ml test tubes and diluted to constant volume， respectively. From each of the test tube， 1 ml was taken and diluted to 5 ml， 1/5 of which， i.e.， 1 ml was taken for later use. The absorbance values of the solutions were determined according to the method of the standard solutions of glucose， and polysaccharide yields were calculated. With the solid-liquid ratio increasing， the polysaccharide yield reached the highest value of 0.86% at the solid-liquid ratio of 1∶11 mg/ml， and then， with the continuous increase of the solid-liquid ratio， the polysaccharide yield decreased， then increased and decreased again. Such results are due to that too-little solvent could not dissolve all the S. nudus， i.e.， soluble polysaccharide could not thoroughly run out， but when the solvent is too much， the concentration is too low， which affects polysaccharide yield. Therefore， the optimal solid-liquid ratio was determined to be 1∶11 mg/ml. Effect of ultrasonic extraction temperature on the extraction rate of S. nudus polysaccharide
As shown in Fig. 5， the crude polysaccharide products of the various experiment groups were dissolved with distilled water， and transferred to 10 ml test tubes and diluted to constant volume， respectively. From each of the test tube， 1 ml was taken and diluted to 5 ml， 1/5 of which， i.e.， 1 ml was taken for later use. The absorbance values of the solutions were determined according to the method of the standard solutions of glucose， and polysaccharide yields were calculated. With the increase of temperature， the polysaccharide also increased， and reached its maximum value of 0.38% at 60 ℃， beyond which the extraction rate decreased with the continuous increase of temperature. Such results are due to that for ultrasonic treatment， the temperature should not be too high， and a too-high temperature would cause the degradation of polysaccharide[10-12]. The optimal extraction temperature was determined to be 60 ℃.
Effect of ultrasonic extraction time on the extraction rate of S. nudus polysaccharide
As shown in Fig. 6， the crude polysaccharide products of the various experiment groups were dissolved with distilled water， and transferred to 10 ml test tubes and diluted to constant volume， respectively. From each of the test tube， 1 ml was taken and diluted to 5 ml， 1/5 of which， i.e.， 1 ml was taken for later use. The absorbance values of the solutions were determined according to the method of the standard solutions of glucose， and polysaccharide yields were calculated. At first， the polysaccharide yield increased over time， and reached its maximum value of 0.31% at 1.5 h， and afterwards， the value decreased over time instead. Such results are due to that ultrasound has stronger mechanical cutting action， and long time action would destruct polysaccharide， causing loss of polysaccharide， which further affects polysaccharide yield[13-17]. The optimal ultrasonic extraction time was determined as 1.5 h.
Effect of ultrasonic extraction times on the extraction rate of S. nudus polysaccharide
As shown in Fig. 7， the crude polysaccharide products of the various experiment groups were dissolved with distilled water， and transferred to 10 ml test tubes and diluted to constant volume， respectively. From each of the test tube， 1 ml was taken and diluted to 5 ml， 1/5 of which， i.e.， 1 ml was taken for later use. The absorbance values of the solutions were determined according to the method of the standard solutions of glucose， and polysaccharide yields were calculated. At first， with the increase of ultrasonic extraction times， the polysaccharide yield increased and reached its maximum value of 1.19% under three times of extraction， and then， the yield decreased with the extraction times increasing instead. Such results are due to that ultrasound has the effect of breaking cells by shaking， and after extracting more than three times， other substances effuse， which affects the yield of polysaccharide. Effect of lye concentration on the extraction rate of S. nudus polysaccharide
As shown in Fig. 8， the crude polysaccharide products of the various experiment groups were prepared and determined according to the experimental operations for polysaccharide solution preparation and determination， and polysaccharide yields were calculated. At first， with the lye concentration increasing， the polysaccharide yield increased and reached its maximum value at the lye concentration of 5%， beyond which the yield decreased with the continuous increase of lye concentration. The results are due to that a too-high lye concentration would degrade polysaccharide， which further reduces the extraction rate. It could be seen that the optimal lye concentration was 5%.
Univariate factors were investigated by single-factor variable method， and the optimal extraction conditions for S. nudus polysaccharide were as follows： solid-liquid ratio at 1∶11 mg/ml， extraction temperature at 60 ℃， three times of ultrasonic extraction， ultrasonic power of 960 W， extraction time of 1.5 h， and lye concentration of 5%， under which the polysaccharide yield was 3.21%.
Determination of antioxidant activity of S. nudus polysaccharide
S. nudus polysaccharide activity of scavenging hydroxyl radical
Determination of hydroxyl radical scavenging capacity of S. nudus polysaccharide
A proper amount of S. nudus polysaccharide was transferred into a dry 100.00 ml beaker， and 0.120 0 g of S. nudus polysaccharide was accurately weighed with an electronic analytical balance. Into the beaker， 10.00 ml of anhydrous ethanol was added， to dissolve all the S. nudus polysaccharide. The solution was transferred into a 100.00 ml volumetric flask， the beaker was flushed with 10.00 ml of anhydrous ethanol for at least 5-6 times， and the flushing fluid was also poured into the 100.00 ml volumetric flask. Then， anhydrous ethanol was added into the volumetric flask to constant volume， obtaining 1.20 mg/ml S. nudus polysaccharide ethanol solution. Next， the 1.20 mg/ml S. nudus polysaccharide ethanol solution was diluted sequentially， obtaining 0.20， 0.40， 0.60， 0.80 and 1.00 mg/ml S. nudus polysaccharide ethanol solutions. Into 6 marked 100 ml conical flasks， 2.00 ml of the different concentrations of S. nudus polysaccharide ethanol solutions were added， respectively， and next， 1.00 ml of 0.010 0 mol/L FeSO4 solution and 1.50 ml of 10% hydrogen peroxide were then added sequentially. After continuous shaking and 10 min of standing， 2.00 ml of 0.0100 mol/L salicylic acid was added to the 6 conical flasks sequentially， and the solutions were stood for 30 min for later use. Each group of the testing samples were scanned with an ultraviolet spectrophotometer， and the optimal wavelength was 510 nm for all of them. Then， at the wavelength of 510 nm， different concentrations of S. nudus polysaccharide ethanol solutions were determined for absorbance A1. According to the same method， into 6 marked 100 ml conical flasks， 2.00 ml of the different concentrations of S. nudus polysaccharide ethanol solutions were added， respectively， and next， 1.00 ml of 0.010 0 mol/L FeSO4 solution and 1.50 ml of distilled water were added sequentially. After continuous shaking and 10 min of standing， 2.00 ml of 0.010 0 mol/L salicylic acid was added to the 6 conical flasks sequentially， and the solutions were stood for 30 min for later use. The solutions were determined with the ultraviolet spectrometer at 510 nm sequentially， for absorbance A2. Also， into the third group of 6 marked 100 ml conical flasks， 2.00 ml of anhydrous ethanol was added instead of S. nudus polysaccharide ethanol solutions， respectively， and next， 1.00 ml of 0.010 0 mol/L FeSO4 solution and 1.50 ml of distilled water were added sequentially. After continuous shaking and 10 min of standing， 2.00 ml of 0.010 0 mol/L salicylic acid was added to the 6 conical flasks sequentially， and the solutions were stood for 30 min for later use. The solutions were determined at 510 nm by the same determination method， for absorbance A0. Meanwhile， with TBHQ as positive control， check test was carried out according to above steps， and the determined results were compared with the results of S. nudus polysaccharide ethanol solutions. Each group of test was designed with three replicates， and the experimental data were recorded and averaged. The hydroxyl radical scavenging rate of S. nudus polysaccharide was calculated according to the computational formula： Hydroxyl radical scavenging rate （%）=[A0- （A1-A2）]/A0×100[18-22]. The determined absorbance data of different concentrations of S. nudus polysaccharide to hydroxyl radical under different conditions were determined， as shown in Table 1. The hydroxyl radical scavenging rates of different concentrations of S. nudus polysaccharide and TBHQ were determined. It could be seen from Fig. 9 that the hydroxyl radical scavenging rate of S. nudus polysaccharide was remarkably lower than common antioxidant TBHQ， and increased with the increase of concentration， basically exhibiting linear relation. At the concentration of 0.20 mg/ml， the hydroxyl radical scavenging rates of S. nudus polysaccharide and TBHQ reached 12.58% and 31.09%， respectively， and hydroxyl radical scavenging rates of both of them increased with the increase of concentration. The regression equation of S. nudus polysaccharide was Y=24.290X+12.915 （R2=0.863 0）， and the regression equation of TBHQ was Y=48.826X+31.939 （R2=0.850 7）. The hydroxyl radical scavenging capacity of S. nudus polysaccharide was about 41%-50% of that of TBHQ， indicating that S. nudus polysaccharide also had very strong hydroxyl radical scavenging capacity， which is relatively lower than common antioxidant TBHQ.
DPPH radical scavenging activity of S. nudus polysaccharide
Determination of DPPH radical scavenging capacity of S. nudus polysaccharide
A proper amount of S. nudus polysaccharide was transferred into a dry 100.00 ml beaker， and 0.120 0 g of S. nudus polysaccharide was accurately weighed with an electronic analytical balance. Into the beaker， 10.00 ml of anhydrous ethanol was added， to dissolve all the S. nudus polysaccharide. The solution was transferred into a 100.00 ml volumetric flask， the beaker was flushed with 10.00 ml of anhydrous ethanol for at least 5-6 times， and the flushing fluid was also poured into the 100.00 ml volumetric flask. Then， anhydrous ethanol was added into the volumetric flask to constant volume， obtaining 1.20 mg/ml S. nudus polysaccharide ethanol solution. Next， the 1.20 mg/ml S. nudus polysaccharide ethanol solution was diluted sequentially， obtaining 0.20， 0.40， 0.60， 0.80 and 1.00 mg/ml S. nudus polysaccharide ethanol solutions. Into 6 marked 100 ml conical flasks， 2.00 ml of the different concentrations of S. nudus polysaccharide ethanol solutions were added， respectively， and next， 2.00 ml of 0.017 8 mol/L DPPH anhydrous ethanol solution was then added sequentially. The solutions were heated in water bath at 40 ℃ for 30 min， for later use. Each group of the testing samples were scanned with an ultraviolet spectrophotometer， and the optimal wavelength was 517 nm for all of them. Then， at the wavelength of 517 nm， different concentrations of S. nudus polysaccharide ethanol solutions were determined for absorbance A1. According to the same method， into other 6 marked 100 ml conical flasks， 2.00 ml of the different concentrations of S. nudus polysaccharide ethanol solutions were added， respectively， and next， 2.00 ml of anhydrous ethanol was then added sequentially. The solutions were heated in water bath at 40 ℃ for 30 min， and determined with the ultraviolet spectrometer at 517 nm for absorbance A2. Also， into the third group of 6 marked 100 ml conical flasks， 2.00 ml of anhydrous ethanol was added instead of S. nudus polysaccharide ethanol solutions， respectively， and next， 2.00 ml of 0.017 8 mol/L DPPH anhydrous ethanol solution was then added sequentially. The solutions were heated in water bath at 40 ℃ for 30 min， and determined with the ultraviolet spectrometer at 517 nm for absorbance A0. Meanwhile， with TBHQ as positive control， check test was carried out according to above steps， and the determined results were compared with the results of S. nudus polysaccharide ethanol solutions. Each group of test was designed with three replicates， and the experimental data were recorded and averaged. The DPPH radical scavenging rate of S. nudus polysaccharide was calculated according to the computational formula： DPPH radical scavenging rate （%）=[A0- （A1-A2）]/A0×100[18-22]. The determined absorbance data of different concentrations of S. nudus polysaccharide to DPPH radical under different conditions were determined， as shown in Table 2. DPPH radical scavenging activity of S. nudus polysaccharide
The DPPH scavenging rates of different concentrations of S. nudus polysaccharide and TBHQ were determined. It could be seen from Fig. 10 that the DPPH scavenging rate of S. nudus polysaccharide was remarkably lower than common antioxidant TBHQ， and increased with the increase of concentration， basically exhibiting linear relation. At the concentration of 0.20 mg/ml， the DPPH scavenging rates of S. nudus polysaccharide and TBHQ reached 12.58% and 32.78%， respectively， and DPPH scavenging rates of both of them increased with the increase of concentration. The regression equation of S. nudus polysaccharide was Y=28.551X+8.080 7 （R2=0.934 7）， and the regression equation of TBHQ was Y=61.633X+26.089 （R2=0.943 1）. The DPPH scavenging capacity of S. nudus polysaccharide was about 37%-44% of that of TBHQ， indicating that S. nudus polysaccharide also had very strong DPPH scavenging capacity， which is relatively lower than common antioxidant TBHQ.
Conclusions
Univariate factors were investigated by single factor variable method， and the optimal extraction conditions for S. nudus polysaccharide were as follows： solid-liquid ratio at 1∶11 mg/ml， extraction temperature at 60 ℃， three times of ultrasonic extraction， ultrasonic power of 960 W， extraction time of 1.5 h， and lye concentration of 5%， under which the polysaccharide yield was 3.21%. The scavenging activity of S. nudus polysaccharide against hydroxyl radical and DPPH radical under different concentrations was determined. At the concentration of 0.20 mg/ml， scavenging rates of S. nudus polysaccharide against hydroxyl radical and DPPH radical were both 12.58%， and the scavenging rates of both the two kinds of radicals increased with the increase of concentration. Through the comparison with the antioxidant activity of antioxidant TBHQ， the antioxidant scavenging capacity of S. nudus polysaccharide was studied in vitro. The hydroxyl radical and DPPH radical scavenging capacities of S. nudus polysaccharide exhibited a remarkable dose-effect relationship， and compared with antioxidant TBHQ at the same concentration， its scavenging capacities of above two kinds of radicals were lower than TBHQ， equivalent to 40%-50% of the effects of TBHQ， indicating that S. nudus polysaccharide has antioxidant activity， with very good effect， and it is directly demonstrated that the radical scavenging capacity of S. nudus polysaccharide is related to the contents of antioxidant components contained in it. S. nudus polysaccharide contains rich aromatics， unsaturated fatty acids， aldehydes， esters and alkenes， all of which have very strong reducibility and might be related to radical scavenging activity. The mechanism of the main functional components with radical scavenging activity still needs further study.