1 Introduction

Enteromorpha prolifera is one of the most common fouling green algae, which distributed worldwide from the intertidal to the upper subtidal zones. It possesses high nutrient value and therapeutical properties (Zhao et al.,2011). Enteromorpha polysaccharide, a group of sulfated heteropolysaccharides, is one of the main biologically active substances in Enteromorphaprolifera. It possessed various bioactive functions, such as blood lipid reduction,immunity, antitumor, and anti-inflammatory (Jiao et al.,2010; Teng et al., 2013; Zhang et al., 2013). However, the high molecular weight and high viscosity limited its pharmaceutical application. Therefore, it was necessary to obtain new pharmacological agents by degrading the Enteromorpha polysaccharide to improve the activity (Xu et al., 2015; Zhang et al., 2013). Among the methods of polysaccharides degradation, oxidation method was used more and more recently (Zhang et al., 2014). To our knowledge, no paper had reported Enteromorphapolysaccharide was degraded by H2O2-Vc method.

Selenium (Se) was a very important essential trace element for biological systems and has been used to nourishment and medicine (Ferri et al., 2003). Recently,selenium polysaccharides were attracting more and more attention due to their excellent high biological activity and low toxicity. It possessed more or stronger biological activities in comparison with selenium-free polysaccharide (Hu et al., 2010). It was also more easily absorbed by organism (Liu et al., 2015; Zhao et al., 2013). Thus, the selenylation modification became intriguing focus in the polysaccharide research field. However, little information has been obtained regarding selenylation modification of the degraded polysaccharide (Lv et al., 2014). In this paper, H2O2–Vc method was introduced to degrade polysaccharide from Enteromorpha prolifera. The antioxidant,antifungal, and antibacterial activities were investigated.It was expected that this investigation would provide encouragement for further exploration into selenylation modification of degraded polysaccharide.

2 Materials and Methods

2.1 Materials

Enteromorpha prolifera was obtained from Qingdao Huiquan waters. Hydrogen peroxide, ascorbic acid (Vc),salicylic acid, ferrous sulfate, sodium selenite, anhydrous ethanol, methanol, 1,1-diphenyl-2-picryl-hydrazyl (DPPH),H2O2 and other reagents were of analytical grade. Dialysis membrane was produced by Spectrum Co., and molecular weight was cut off at 500 Da. Diethylaminoethylcellulose (DEAE-52) was purchased from Beijing Leybold Cable Technology Co., Ltd.

Cytospora sp., Apple anthrax, Fusarium oxysporum vasinfectum, Colletotrichum capsici, Alternaria brassicae,Staphlococcus aureau, Eschetichia coli, Bacillus pumilus,Bacillus subtilis, Salmonella, Micrococcus luteus were provided by Bionic Test Center in Qingdao Agricultural University.

2.2 Analytical Methods

2.2.1 Chemical analysis

由于BCD-I节点获取了凸包H的信息,BCD-I节点就利用空洞核心信息HCI(Hole Core Information)消息向邻居节点传输凸包H信息。一旦接收到HCI消息,节点就计算凸包H的视图控制角θ。如果θ大于αmin,就存储凸包Ω的信息,再将HCI消息传输至它的邻居节点;否则就丢弃HCI消息。

2.2.2 Structure analysis

UV spectra were measured by shimadzu UV 2550 from 200 to 400 nm. Fourier transform infrared (FT-IR) spectra were measured by Thermo Scientific Nicolet iS10 FT-IR spectrometer (Waltham, MA) in KBr disks from 500 to 4000 cm−1.

2.3 Preparation of Degraded Polysaccharide (LEP)and Degraded Polysaccharide Selenide (Se-LEP)

The extraction and purification of polysaccharide (EP)from Enteromorpha prolifera was performed according to the method that we previously reported (Lü et al. 2013).

3.3.1 IR spectroscopic analysis

Three kinds of degradation reagent were investigated to achieve efficient degradation of polysaccharide, (1) 5 mL 10 mmol L−1 H2O2, (2) 5 mL 10 mmol L−1 ascorbic acid ,(3) 5 mL 10 mmol L−1 H2O2 and 5 mL 10 mmol L−1 ascorbic acid. The antioxidant activity was used to evaluate the effect of degradation by DPPH free radical scavenging assay (Lv et al., 2014). Since the antioxidant activity of the purified polysaccharide was often evaluated in vitro by hydroxyl free radical (·OH) scavenging assay, ·OH was chosen as evaluating the effects of degradation.

2.3.2 Optimizing preparation of low-molecularweight polysaccharide (LEP)

Polysaccharides have shown many biological activities,such as antitumor, antioxidation, immune-stimulating,and neuroprotecting effects. Degradation of polysaccharide into LEP was expected to improve its biological activity. In this paper, the antioxidant activity of LEP wasused to evaluate the effect of degradation by DPPH free radical scavenging assay. LEP was obtained by varying reaction time, reaction temperature, H2O2/Vc (n/n=1:1)concentration, and solid-liquid ratio. An orthogonal design L9 (34) was adopted to select the optimum experimental conditions (shown in Table 1). 0.1 g polysaccharide was used to prepare LEP in each experiment. The values for different variables were chosen by the single factor experiments.

基体元素信号值远远高于其空白值，且基体元素信号值的稳定性也是影响测定结果稳定性的主要因素，故实验选择63Cu的信号强度为考核对象。在表1中其他条件参数不变的情况下，采用纯铜光谱标准样品T02逐级优化了激光剥蚀系统的激光能量、剥蚀孔径、扫描速率以及载气流速4个影响激光剥蚀固体进样产生瞬时信号的关键参数，使63Cu的信号强度稳定并达到最大值[11]。

Table 1 Orthogonal experimental results for the preparation of LEP

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2.3 .3 Preparation of degraded polysaccharide selenide (Se-LEP)

The preparation of Se-LEP was performed according to the method that we previously reported (Lü et al., 2014).Briefly, 0.1 g LEP was put into a 50 mL round-bottomed flask, to which 10 mL 1.0% nitric acid solution and 0.1 g sodium selenite were added. The mixture was reacted at 60℃ for 5 h. After centrifuged, the residual sodium selenite in the supernatant was dialyzed with distilled water.And then vacuum freeze-dried, Se-LEP was obtained.

2.4 Antioxidant Activity Assay

2.4.1 Scavenging activity of DPPH radicals

2 mL 0.1 mmol L−1 DPPH was added to 1 mL various doses of polysaccharide (0.2, 0.4, 0.6, 0.8 and 1 mg mL−1)in 10 mL test tubes, respectively. The mixture was shaken vigorously, and kept at room temperature for 30 min.Then, the absorbance was obtained at 517 nm by UV-visible spectrophotometer. The DPPH radical scavenging activity was calculated as:

2.4.2 Scavenging activity of hydroxyl radical

where SEDPPH was DPPH scavenging effect, A0 was the absorbance of the blank group (distilled water + DPPH),A1 was the absorbance of the sample reaction (sample +DPPH), and A2 was the background absorbance of the sample (distilled water replaced DPPH).

数学的教学，不仅仅是数学思维的激荡，还当是数学活动的碰撞。动手操作是初中数学教学中的基本形式，无论是数字和方程的运算，还是图形的解构和模型的制作，都离不开学生的动手操作。在教学中，教师可以在普遍而又零散的动手操作习惯的基础上，组织专项的动手操作活动，以有效地培养学生的探究实践意识和能力。

where SEhydroxyl was hydroxyl scavenging effect, A1 was the absorbance of the sample, A2 was the background absorbance of the sample, A3 was the absorbance of blank.

2 mL various doses of polysaccharide (0.25, 0.5, 1.0,2.0, 4.0 mg mL−1) in 10 mL test tube were mixed with 1 mL FeSO4 (9 mmol L−1) and 1 mL salicylicacid-ethanol solution (9 mmol L−1), respectively. Then, 1 mL H2O2 (8.8 mmol L−1) was added before incubation at 37 for 60℃min. The hydroxyl radical scavenging activity was calculated as:

(1)对原始矩阵DSM和DPM/MIM分别进行预排序，使得各集合Li中的构件聚集在一起。对于图2所示的模型，若C2,C4和C5必须放置在同一模块，C6和C8必须放置在同一模块中，则一种构件序列为(C1,C3,C2,C4,C5,C6,C8,C7,C9,C10,C11)。预排序的目的是为了构造算法的搜索算子。

②使用四次复合数据进行验证,验证误差结果如下:Fx方向最大误差为69.74%,Fy方向最大误差为230.40%,Fz方向最大误差为240.81%,Mx方向最大误差为64.49%,My方向最大误差为133.58%,Mz方向最大误差为124.65%,显然使用单维数据训练得到的网络无法对多维复合数据进行解算。

The antioxidant activity assays were performed in triplicate and results are expressed as mean values ± standard deviations (SD).

2.5 Antifungal and Antibacterial Activities

Staphlococcus aureau, Eschetichia coli, Bacillus pumilus, Bacillus subtilis, Salmonella, and Micrococcus luteus were cultured in LB/MH broth at 37 ℃, which were used to test the antifungal activities. Cytospora sp., Apple anthrax, Fusarium oxysporum vasinfectum, Colletotrichum capsici, and Alternaria brassicae were cultured in PDA broth at 28 ℃, which were used to test the antibacterial activities.

The minimal inhibitory concentration (MIC) values were determined based on a micro-well dilution method as previously described with modifications (Sarker et al.,2007). The 96-well plates were prepared by dispensing,into each well, 5 μL of sample, and 95 μL of the inoculants in nutrient broth. After incubation under aerobic conditions for 24 h at 37℃ for bacteria, and 48 h at 28℃for fungus, MIC was defined as the lowest concentration of sample at which no visible growth could be detected.Sample free solutions were used as blank controls. The antifungal and antibacterial activity assays were performed in triplicate.

3 Results and Discussion

3.1 Selection of Degradation Reagent

When H2O2, Vc, and H2O2/Vc were tested as the degradation reagent, their extraction rates of degraded polysaccharide were 76.30%, 77.10%, and 73.10%, and their scavenging rates of hydroxyl free radical were 41.16%,40.22% and 56.35%, respectively. H2O2/Vc was the best degradation reagent.

3.2 Preparation of LEP and Se-LEP

LEP was prepared by varying reaction time, reaction temperature, H2O2/Vc (n/n=1:1) concentration, and solidliquid ratio. The scavenging rate varied from 29.58% to 61.58%. According to the statistical analysis, the optimum conditions of LEP were reaction time of 2 h, reaction temperature of 50℃, H2O2/Vc (n/n=1:1) concentration of 15 mmol L−1, and solid-liquid ratio of 1:50 (g mL−1). Under these conditions, the scavenging rate was 64.71%, which was higher than those in Table 1. The yield of LEP was 58.33%. The selenium content of Se-LEP was 1137.29 μg g−1, while the selenium content in Se-EP was only 493.5 μg g−1. The content of sulfate radical was 22.44%, 22.82%, 22.84% and 22.96% for EP, Se-EP, LEP, and Se-LEP, respectively. After degradation or selenylation, the content of sulfate radical has no change.The bioactivities of sulfate radical in different polysaccharide did not change.

知识体系的建立在对学科知识的发展规律充分认知的基础上，由对编辑出版学与其他学科知识勾连的梳理，到形成核心问题群和明确核心概念，继而进入学科建构的核心环节即编辑出版学学科知识体系的建立。从三个方面看，知识体系是核心，课程体系是路径，而人才培养体系是学科生命在制度化的过程中得以延续的关键，三者实现了逻辑、方法和效果的统一。

3.3 Structure Spectroscopic Analysis

From the UV spectra, there was no absorption at 280 and 260 nm, which indicated protein and nucleic acid were not observed and their contents were very low.There was no absorption appeared at 210 nm, indicating the absence of SeO32−. However, there was a strong absorption at 196.50 nm for Se-LEP, and no absorption for LEP. Thus, selenite ester groups might be formed in Se-LEP, which demonstrated further by IR.

Total sugar content was analyzed with the phenol-sulfuric acid method. Selenium content analysis was performed by graphite furnace atomic absorption spectrometer WFX-210. The content of sulfate radical was analyzed with the reported method (Xiong et al., 2013).Molecular weight was determined by Waters 1525 Gel Permeation Chromatography system with refractive index detector.

2.3.1 Selection of degradation reagent

Fig.1 IR spectra of EP, LEP and Se-LEP.

The wide and strong absorption peak at 3430.62 cm−1 corresponded to the stretching vibrations of hydrogen bonded OH groups. The band at around 2946.57 cm−1 was C-H stretching vibration. The bands attributed to C=O stretching vibrations appeared at 1635.1 cm−1, which indicated the existence of uronic acids. The bands attributed to C-O stretching vibrations appeared at 1417.41 cm−1.The bands attributed to S=O stretching vibrations at 1226.58 cm−1, and the bands attributed to C-O-S stretch ing vibrations at 849.70 cm−1, which indicated the existence of sulfate radical. The bands attributed to C-O-C stretching vibrations of at 1070.60 cm−1, which indicated the existence of pyranose ring. The FT-IR spectroscopy of Se-LEP presented one characteristic absorption band at 712.51 cm−1 describing stretching vibration of Se=O. As compared with the spectrogram of EP and LEP, Se-LEP was succeeded in selenylation modification. The conjugation of selenium did not affect the main structure of the polysaccharide.

3.3.2 Molecular weight analysis

The molecular weight determination was measured by high-performance gel permeation chromatography (HPGPC). Samples (10.0 mg) were dissolved in distilled water(10.0 mL), passed through a 0.45 μm filter and applied to a gel-filtration chromatographic column of Ultrahydrogel TM Linear (300 mm×7.8 mm, Waters, USA). 3 mmol L−1 sodium acetate was used as the flow phase at a flow rate of 0.8 mL min−1. The temperature of the column was maintained at 25℃ and the injection volume was 20 μL.Preliminary calibration of the column was carried out using glucan standards with different molecular weights(2500, 4600, 7100, 21400, 41100, 133800 Da). According to the standard curve

where Mw represented relative molecular weight, Rt represented retention time), the average molecular weights of EP and Se-LEP were calculated to be about 8.4×104Da,and 5.4×104Da, respectively.

老陆变化太大了。看上去比你还要老（我老吗？），头发都掉光了，秃脑门儿。上学那会，也算是个帅哥，现在整个一老头模样，简直不敢认了。你知道他是做什么的吗，教育局当处长，你知道他开的什么牌子的车，奥迪Q 5，我操，六七十万吧！

10篇文献报道了多孔钽金属加强块重建Paprosky II、III型髋臼骨缺损导致术区感染的并发症，各研究间不存在统计学异质性（P=0.57，I2=0.0%），采用固定效应模型进行分析。荟萃分析结果显示：术区感染发生率3.59%（95%CI：0.03～0.07），不同文献报告的该并发症发生率差异有统计学意义（图1）。

3.3.3 Separation and purification by DEAE-52 column

Se-LEP was sequentially purified by DEAE-52 column according to the reported methods with minor modifycation (Dubois et al., 1956). The Se-LEP solution (2 mL,20 mg mL−1) was applied to a column (1.6 cm × 50 cm) of DEAE-52 cellulose. Then, the column was stepwise eluted with 0, 0.1, 0.3 and 0.5 mol L−1 NaCl solutions at a flow rate of 0.5 mL min−1. The resulting elute (10 mL/tube)was collected and the polysaccharides were detected at 490 nm by the phenol-sulfuric acid method. Five fractions of Se-LEP were obtained. Each fraction was collected, concentrated, and further purified through a column (1.5 cm×100 cm) of Sephadex G-100, resulting in six purified fractions. Finally, six fractions were lyophilized for further investigation.

Fig.2 Elution curve of Se-LEP on DEAE-52 column.

3.4 Antioxidant Activity in vitro

3.4.1 Scavenging activity of DPPH radical

大丫的美容中心，不仅仅是一般性的豪华，而是金碧辉煌！整个中心是欧式的，到处都是巨大的镜子，人走在这里如同进了琉璃世界。

DPPH was a free radical compound and had been used widely to test the free radical scavenging ability of various samples. With the color of solution changing from purple to yellow, the radical would be scavenged and the absorbance reduced. The DPPH free radical scavenging effects of EP, LEP, Se-EP and Se-LEP were measured and the results were given in Fig.3. It was due to the hydrogen-donating ability that the higher the scavenging rate was for DPPH free radical, the higher antioxidant activity. Scavenging effect of polysaccharide on DPPH radical increased with the increasing of concentration.Among EP, Se-EP, LEP, and Se-LEP, Se-LEP has the strongest scavenging capacity.

朱自清说：“在专制时代的种种社会条件之下，集体的行动是不容易表现的，于是士人的立身处世就偏向了‘节’这个标准。在朝的要做忠臣。这种忠节或是表现在冒犯君主尊严的直谏上，有时因此牺牲性命；或是表现在不做新朝的官甚至以身殉国上。忠而至于死，那是忠而又烈了。在野的要做清高之上，这种人表示不愿和在朝的人合作，因而游离于现实之外，或者更逃避到山林之中，那就是隐逸之士了。”

（1）电流频率 为实现规定深度的高质量的感应加热，首先必须正确选择设备的频率。设备频率除对实现技术要求和提高热处理质量有很大作用外，对于充分发挥设备的效能、提高生产率、节省电能也很重要。所谓频率的选择，这里指的是选择合理的频带或频率范围，并不是严格的具体数值。

Fig.3 Scavenging effects on DPPH radical. Results are expressed as means values ± standard deviation (SD). All assays were performed in triplicate.

3.4.2 Scavenging activity of hydroxyl radical

The hydroxyl radical possessed extremely high reactivity and could induce severe damage to functioning biomolecules in living cells. In principle, this could be prevented and/or inhibited by antioxidants. ·OH was generated by mixing H2O2 and Fe2+, and it had a high reactive behavior but short survival time. If salicylic acid was added, ·OH could be effectively captured, and colored substances were produced. The substances had a strong absorption at 510 nm wavelength. However, when adding an analyte with scavenging function of ·OH in the reaction system, it would compete with salicylic acid for ·OH,which would reduce the generation of the amount of colored substances. Using the method of fixing reaction time,different amounts of analyte in the same volume of reaction system were added to determine the absorbance at 510 nm. In this way, the scavenging capacity of the ana-lyte on ·OH could be determined. Fig.4 showed EP,Se-EP, LEP and Se-LEP had strong scavenging effect on ·OH, and the scavenging capacity was proportional to the quantity of materials, and at the same conditions,Se-LEP had the strongest scavenging capacity.

These results indicated that degradation polysaccharides should be explored as potential antioxidants.

Fig.4 Scavenging effects on hydroxyl radical. Results are expressed as means values ± standard deviation (SD). All assays were performed in triplicate.

3.5 Antifungal and Antibacterial Activities

Under the experimental concentrations of 50 mg mL−1,the antibacterial and antifungal effects of EP, Se-EP and LEP were not obvious. However, Se-LEP showed significant antibacterial properties for Escherichia coli, Bacillus subtilis and Salmonella spp. with the MIC of 50, 25,25 mg mL−1, respectively. Se-LEP showed significant antifungal properties for Apple anthrax with the MIC of 25 mg mL−1.

The antifungal and antibacterial activities results established the antifungal and antibacterial potency of Se-LEP.However, the structure of Se-LEP was very complex, and its biological activity was usually not a function of one single factor but rather a combination of factors. The relationship between the structure of Se-LEP and antifungal,antibacterial mechanism required further studies.

4 Conclusions

In the present study, an efficient degradation method of Enteromorpha polysaccharide by free radicals method with H2O2 and Vc was developed. Se-LEP was synthesized with high Se content, while the content of sulfate radical did not change. Se-LEP had significant antioxidant, antifungal, and antibacterial activities. However, the mechanism of antioxidant activities in vivo and the safety of sulfated polysaccharides from Enteromorphaprolifera for human metabolism should be further researched.

Acknowledgements

This research was financially supported by the National Key R&D Program of China (No. 2017YFD0501500),and the National Key Technology R & D Program of China (No. 2015BAD11B01-04).

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