马尾藻多糖的分离纯化、生物活性及结构分析
|
摘要
马尾藻(Sargassum pallidum)属褐藻门马尾藻科植物,盛产于我国黄海、渤海沿岸各地,资源十分丰富,由于不能直接食用,以致目前马尾藻的加工利用范围比较窄,只有小部分被用作饲料、藻胶和医药工业的原料,大部分尚未得到全面的开发利用。褐藻糖胶(褐藻多糖)是褐藻中的一种杂多糖,具有多样的生理功能,是褐藻中重要的生物活性物质。目前国内外对马尾藻中的多糖(S.pallidum Polysaccharides,SPP)研究很少,且不够深入。鉴于此,本文对采自山东荣成的马尾藻中多糖的体外抗肿瘤活性与体外抗氧化活性,以及多糖的结构进行了研究,以期了解它们的生理活性与结构。
本论文共有四个部分,分别对马尾藻多糖的提取、分离纯化和理化性质、体外抗肿瘤活性与抗氧化活性、结构鉴定进行了研究。主要结果如下:
1马尾藻多糖的提取
利用超临界二氧化碳(SC-CO_2)脱除马尾藻粉中的脂类物质,研究了超临界CO_2萃取压力、萃取温度、萃取时间对马尾藻脱脂过程的影响。研究结果表明CO_2的流量为20 L/h时,超临界CO_2脱脂的最佳工艺条件为:萃取压力45 MPa,萃取温度55℃,时间6 h,萃取率为8.53‰。
将响应面分析法用于马尾藻多糖的提取工艺,对影响马尾藻多糖提取的主要因素超声波破碎时间、提取温度、提取时间的最佳水平范围及其交互作用进行了研究和探讨,依据回归分析结果,最佳工艺条件是:超声波破碎时间为469.54 s,提取温度为90.94℃,提取时间为4.96 h,多糖得率的理论值为2.48%。对三氯乙酸(TCA)脱蛋白条件进行了优化,选择TCA终浓度为2%左右,TCA沉淀蛋白的时间为2h或稍长。
在压力0.5 MPa,温度20℃下,对脱蛋白后的提取液进行膜分离,提取液依次用孔径0.1μm的微滤膜、截留分子量为100 KDa、50 KDa、10 KDa和3 KDa的超滤膜进行膜分离。研究结果表明多糖组分主要包含在分子量大于50 KDa的截流液中,由于大于孔径0.1μm的截流溶液中所含多糖量较多,成分较复杂,将其单独收集分析。分子量小于50 KDa的溶液多糖含量较少,将其合并。在以后的实验中将SPP提取液分成三部分SPP-1(＞0.1μm)、SPP-2(＜0.1μm且＞50 KDa)和SPP-3(＜50 KDa)。
研究了马尾藻料液浓度对膜分离的影响,在料液比分别为1:20(m/V)、1:30、1:100、1:150,压力为0.5 MPa,温度为20℃时,将脱蛋白后的提取液进行膜分离,结果表明随着料液比的增加,膜分离后的多糖损失越少。综合考虑,在膜分离前将待处理的溶液按1:150稀释。在温度20℃时,压力分别在0.1、0.3、0.5 MPa下进行膜分离,结果表明操作压力对多糖损失并无显著影响,在膜分离设备允许压力范围内,0.1-0.5 MPa都是适合的。
2马尾藻多糖的纯化
多糖组分SPP-1、SPP-2和SPP-3占粗多糖百分比分别为72.15%、16.69%和11.16%,其中的多糖含量依次为58.13%、18.98%和16.38%,蛋白含量依次为1.09%、1.75%和1.73%,硫酸基含量依次为15.68%、15.26%和22.62%,糖醛酸含量依次为14.22%、14.30%和5.20%。
利用DEAE Cellulose-52层析柱分别对SPP-1、SPP-2和SPP-3三个多糖组分进行分离纯化,其中由SPP-1得到两个主要的组分SPP-1-1和SPP-1-2,得率分别为32.53%和8.67%;由SPP-2得到SPP-2-1、SPP-2-2和SPP-2-3三个组分,得率分别为10.53%、9.20%和36.33%;由SPP-3得到SPP-3-1和SPP-3-2两个组分,得率分别19.52%和57.27%。在纯化过程中多糖有部分损失,同时粗多糖中的蛋白基本被除去。利用Sephadex G-100层析柱分别对SPP-1-1、SPP-1-2、SPP-2-1、SPP-2-2、SPP-2-3、SPP-3-1和SPP-3-2七个组分进行分离纯化,以用于进一步的结构分析。
3马尾藻多糖的体外抗肿瘤活性与抗氧化活性
以MTT比色法探讨了经DEAE Cellulose-52纯化得到的七个多糖组分对人肺癌细胞株A549、人肝癌细胞株HepG2和人胃癌细胞株MGC-803生长的体外抑制作用。组分SPP-3-1和SPP-3-2对人肝癌细胞株HepG2、人肺癌细胞株A549和人胃癌细胞株MGC-803的生长具有明显的体外抑制作用,且抑制能力具有剂量依赖性。组分SPP-2-3只对肝癌HepG2细胞表现出强抑制率,并呈剂量依赖性。组分SPP-3-1和SPP-3-2具有较高的体外抗肿瘤活性可能与它们的低分子量(＜50 KDa)及高硫酸基含量(22.62%)有关。
在浓度为2 mg/mL时,粗多糖组分SPP-1、SPP-2和SPP-3对DPPH自由基的清除率分别为23.84%、13.23%和12.83%;总抗氧化能力分别为0.087、0.083和0.088(μmolFeSO_4/mg);还原力(以700 nm处的吸光度A_(700)表示)分别为0.785、0.446和0.541;对·OH的清除率分别为68.39%、13.22%和27.59%。
研究了乙醇浸提温度对马尾藻中总多酚含量的影响,在30℃马尾藻中的多酚类物质不能完全浸提出来;在70℃因浸出时间短不易控制,所以选择50℃作为乙醇浸提温度。马尾藻经50℃、70%乙醇浸提后,依次用不同有机溶剂萃取后得到乙酸乙酯萃取物、正丁醇萃取物及萃取剩余物,其占总萃取物百分比依次为15.76%、5.28%、78.96%,采用Folin-Ciocalteu比色法测定其中的总多酚含量,以绿原酸(CHA)作为标准物质,依次为3.20 mgCHA/g干重、12.12 mgCHA/g干重和52.08 mgCHA/g干重,总萃取物得率为8.07%。
乙酸乙酯萃取物和正丁醇萃取物在浓度为2 mg/mL时对DPPH自由基清除率分别为30.59%和29.36%。乙酸乙酯萃取物和正丁醇萃取物的总抗氧化能力较高,分别为0.520和0.345(μmolFeSO_4/mg)。乙酸乙酯萃取物、正丁醇萃取物和萃取剩余物的还原力较高,由高到低为萃取剩余物＞正丁醇萃取物＞乙酸乙酯萃取物。乙酸乙酯萃取物、正丁醇萃取物和萃取剩余物在浓度为2 mg/mL时对金属离子的螯合率分别为48.25%、51.34%和17.21%。
4马尾藻活性多糖的结构分析
利用高效凝胶过滤色谱法(HPGFC)测定多糖组分SPP-2-3、SPP-3-1、SPP-3-2的分子量和检测其纯度,其平均分子量(M_w)依次为132.999 KDa、5.874 KDa、7.249KDa。气相色谱法分析多糖的单糖组成,SPP-2-3单糖组成及摩尔比为鼠李糖:木糖:岩藻糖:甘露糖:葡萄糖:半乳糖=0.15:2.74:0.64:1.00:0.49:1.11,SPP-3-1单糖组成及摩尔比为甘露糖:葡萄糖:半乳糖=1.00:11.18:0.96,SPP-3-2单糖组成及摩尔比为鼠李糖:木糖:岩藻糖:甘露糖:葡萄糖:半乳糖=0.17:2.53:0.61:1.00:0.46:0.92。通过红外吸收光谱(IR)分析得知SPP-2-3和SPP-3-2中存在(?)糖苷键。
高碘酸氧化、Smith降解以及甲基化分析结果显示SPP-2-3中的甘露糖的连接方式以(1→3)糖苷键为主,葡萄糖和半乳糖除了存在(1→3)键型外,还存在(1→6)键连接方式;木糖的连接方式以(1→2)键为主;岩藻糖除了存在(1→3)键型外,还存在(1→4)键型。SPP-3-2中的甘露糖和半乳糖的连接方式以(1→3)键为主;葡萄糖是以(1→6)或者(1→2)键型连接;木糖和岩藻糖以(1→2)或(1→4)键型连接为主。
Sargassum pallidum belongs to Phaeophyta Sargassaceae and are extensively distributed in China Yellow Sea and Bohai Sea.Due to not being edible,S.pallidum is used in relatively narrow scope at present,and only a small part is used as feed,algin and raw materials of pharmaceutical industry,and most of them has not yet been fully utilized.Alga glycine is a kind of miscellaneous polysaccharide.It has a variety of physiological functions,and is an important bioactive substance in brown alga.However,little information about S.pallidum polysaccharides(SPP) and biological activity is available compared with those of the other Sargassum genus at present.In this paper S.pallidum collected from Rongcheng in Shandong Province was studied,including the antitumor activity and antioxidant activity in vitro of SPP,and identification,of polysaccharides structure.
This paper is composed of four parts:extraction of polysaccharides from S.pallidum, purification and identification of physical-chemical characters,antitumor activity and antioxidant activity of SPP in vitro,and identification of polysaccharides structure.Main results are listed as follows:
1.Extraction of S.pallidum polysaccharides
Supercritical CO_2(SC-CO_2) extraction was used to degrease the lipophylic substances in S.Pallidum.The effect of extraction pressure,extraction temperature and extraction time were investigated.As results,the optimum operating conditions for treatment were as follows:temperature at 55℃,pressure at 45 MPa,a flow rate of CO_2 at 20 L/h,and extraction time for 6 h,and the yield was 8.53‰.
Extraction of S.pallidum polysaccharide was optimized by the method of reSPPonse surface analysis(RSA).The effects of the main variables namely:ultrasonic time,extraction temperature and extraction time as well as the mutual interaction between variables were studied.The maximum yield of polysaccharides was up to 2.48%under the conditions as follows:ultrasonic time 469.54 s;extraction temperature 90.94℃;extraction time 4.96 h. The conditions of deproteinization by trichloroaeetie acid(TCA) were optimized.TCA final concentration was about 2%,and precipitation time was 2 h or longer.
Membrane separation was performed at 0.5 MPa and at 20℃.The deproteinized polysaccharides solution was pumped to membrane surface(tangential flow) with different nominal molecular weight cut-offs(0.1μm,100 KDa,50 KDa,10 KDa,3 KDa),in turn. The results showed that S.pallidum polysaccharides were mainly included in the fractions with molecular weight more than 50 KDa.Due to the fraction with pore size more than 0.1μm contained much polysaccharides,having complex composition,it was collected and analyzed singly.Fractions with molecular weight less than 50 KDa containing less polysaccharide were combined.In later experiments S.pallidum polysaccharides(SPP) were divided into 3 main crude polysaccharides,SPP-1(＞0.1μm),SPP-2(＜0.1μm and＞50 KDa) and SPP-3(＜50 KDa).
Effect of the ratio(material to water) on membrane separation was studied.At the ratio(material to water) of 1:20(m/V),1:30,1:100 and 1:150,pressure at 0.5 MPa, temperature at 20℃,the deproteinized polysaccharides solution were treated by membrane separation.The results showed that a higher ratio(material to water) led to a less loss of polysaccharides in membrane separation.It was considered to be suitable to dilute the solution at the ratio of 1:150 before membrane separation.At 20℃,membrane separation was performed at the pressure of 0.1,0.3 and 0.5 MPa,respectively.The results showed that the operating pressure had no significant effect on polysaccharides loss.So the pressure between 0.1 and 0.5 MPa are suitable in the scope allowed by membrane separation equipment.
2.Purification of S.pallidum polysaccharides
The yield of SPP-1,SPP-2 and SPP-3 accounted for 72.15%,16.69%and 11.16%, respectively of total crude polysaccharides.The polysaccharides contents were 58.13%, 18.98%and 16.38%,respectively and the protein contents were 1.09%,1.75%and 1.73%, respectively.The sulfate contents were 15.68%,15.26%and 22.62%,respectively and the uronic acid contents were 14.22%,14.30%and 5.20%,respectively.
A lyophilized fraction of polysaccharides SPP-1 was chromatographed on a DEAE Cellulose-52 anion-exchange column to yield two peaks,SPP-1-1 and SPP-1-2.In a similar manner,SPP-2-1,SPP-2-2 and SPP-2-3,SPP-3-1 and SPP-3-2 were obtained from SPP-2 and SPP-3,respectively.The recovery of SPP-1-1 and SPP-1-2 were 32.53%and 8.67%, respectively.The recovery of SPP-2-1,SPP-2-2 and SPP-2-3 were 10.53%,9.20%and 36.33%,respectively.The recovery of SPP-3-1 and SPP-3-2 were 19.52%and 57.27%, respectively.During the purification of crude polysaccharides a little part of polysaccharides were losed,while the protein in crude polysaccharide was almost removed. Fractions of polysaccharides SPP-1-1,SPP-1-2,SPP-2-1,SPP-2-2,SPP-2-3,SPP-3-1 and SPP-3-2,respectively were chromatographed on a Sephadex G-100 chromatography column.So they could be used for further structural analysis.
3.Antitumor activity and antioxidant activity in vitro of S.pallidum polysaccharides
The inhibition effects of 7 fractions purified by DEAE Cellulose-52 on the growth of HepG2 cells,A549 cells and MGC-803 cells were evaluated in vitro by MTT assay. Fractions of polysaccharides SPP-3-1 and SPP-3-2 presented significantly higher antitumor activity against the HepG2 cells,A549 cells and MGC-803 cells in vitro than blank control groups,and the inhibition ability was dose-dependent.SPP-2-3 only presented significantly higher antitumor activity against the HepG2 cells in vitro than a blank control, dose-dependently.It seems that SPP-3-1 and SPP-3-2 from SPP-3 with relatively lower molecular weight(＜50KDa) and higher sulfate contents(22.62%) showed significantly higher antitumor activity against the HepG2 cells,A549 cells and MGC-803 cells in vitro.
At the concentration of 2mg/mL,scavenging of DPPH radical of crude polysaccharide SPP-1 and SPP-2 and SPP-3 were 23.838%,13.232%and 12.828%,respectively.The total antioxidant capacity of SPP-1 and SPP-2 and SPP-3 were 0.087,0.083 and 0.088, respectively(μmolFeSO4/mg).The reducing power(expressed as absorbanee at 700 nm) and OH radical scavenging of SPP-1,SPP-2 and SPP-3 increased with the increasing concentration in experimental scope.Their reducing power were 0.785,0.446 and 0.541, respectively at 2 mg/mL.Their OH radical scavenging were 68.391%,13.218%and 27.586%,respectively at 2 mg/mL.
Effect of ethanol extraction temperature on total polyphenol content extracted from S. pallidum were studied.At 30℃total polyphenol could not been fully extracted from S. pallidum,while at 70℃the extraction time was too short to be controlled,so 50℃was selected as alcohol extraction temperature.S.pallidum was extracted by 70%ethanol at 50℃,and then extracted by chloroform,ethyl acetate and n-butanol in turn.The yeied of total extract was 8.07%.Ethyl acetate extract,n-butanol extract and extract residue accounted for 15.76%,5.28%and 78.96%,respectively of total extract,and their total polyphenol contents were 3.20 mgCHA /gDW,12.12 mgCHA/gDW and 52.08 mgCHA/gDW, respectively determined by Folin-Ciocalteu method.
DPPH radical scavenging of ethyl acetate and n-butanol extract were 30.59%and 29.36%,respectively at 2 mg/mL.Their total antioxidant capacity were 0.520 and 0.345 (μmolFeSO4/mg),respectively.Reducing power of ethyl acetate extracts,n-butanol extract and extract residue were high.The descending sequence was extract residue,n-butanol extract and ethyl acetate extracts.Metal ions scavenging of ethyl acetate extracts,n-butanol extracts and extract residue were 48.25%,51.34%and 17.21%at 2 mg/mL.
4.Structural analysis of S.pallidum polysaccharides
The average molecular weight and purity of polysaccharides fractions SPP-2-3, SPP-3-1 and SPP-3-2 were defined by high performance of gel filtration chromatography (HPGFC).They were 132.999KDa,5.874KDa and 7.249KDa,respectively. Monosaccharide composition and molar ratio of SPP-2-3,SPP-3-1 and SPP-3-2 were analyzed by Gas chromatography(GC).They were rhamnose:xylose:fucose:mannose: glucose:galactose= 0.15:2.74:0.64:1.00:0.49:1.11,mannose:glucose:galactose= 1.00:11.18:0.96,and rhamnose:xylose:fucose:mannose:glucose:galactose=0.17: 2.53:0.61:1.00:0.46:0.92,respectively.The infrared absorption spectroscopy(IR) of SPP-2-3 and SPP-3-2 showed a character of口-glycosidic bond.
Periodate oxidation,Smith degradation and methylation analysis of SPP-2-3 showed that mannose were linked mainly by(1→3) glycosidic bond.In addition to(1→3) glycosidic bond in glucose and galactose,there was also(1→6) glycosidic bond.Xylose was linked mainly by(1→2) glycosidic bond.In addition to(1→3) glycosidic bond in fucose,there was also(1→4) glycosidic bond.For SPP-3-2,mannose and galactose were linked mainly by(1→3) glycosidic bond.Glucose was linked by(1→6) or(1→2) glycosidic bond.Fucose and L-xylose was linked by(1→2) or(1→4) glycosidic bond.
本论文共有四个部分,分别对马尾藻多糖的提取、分离纯化和理化性质、体外抗肿瘤活性与抗氧化活性、结构鉴定进行了研究。主要结果如下:
1马尾藻多糖的提取
利用超临界二氧化碳(SC-CO_2)脱除马尾藻粉中的脂类物质,研究了超临界CO_2萃取压力、萃取温度、萃取时间对马尾藻脱脂过程的影响。研究结果表明CO_2的流量为20 L/h时,超临界CO_2脱脂的最佳工艺条件为:萃取压力45 MPa,萃取温度55℃,时间6 h,萃取率为8.53‰。
将响应面分析法用于马尾藻多糖的提取工艺,对影响马尾藻多糖提取的主要因素超声波破碎时间、提取温度、提取时间的最佳水平范围及其交互作用进行了研究和探讨,依据回归分析结果,最佳工艺条件是:超声波破碎时间为469.54 s,提取温度为90.94℃,提取时间为4.96 h,多糖得率的理论值为2.48%。对三氯乙酸(TCA)脱蛋白条件进行了优化,选择TCA终浓度为2%左右,TCA沉淀蛋白的时间为2h或稍长。
在压力0.5 MPa,温度20℃下,对脱蛋白后的提取液进行膜分离,提取液依次用孔径0.1μm的微滤膜、截留分子量为100 KDa、50 KDa、10 KDa和3 KDa的超滤膜进行膜分离。研究结果表明多糖组分主要包含在分子量大于50 KDa的截流液中,由于大于孔径0.1μm的截流溶液中所含多糖量较多,成分较复杂,将其单独收集分析。分子量小于50 KDa的溶液多糖含量较少,将其合并。在以后的实验中将SPP提取液分成三部分SPP-1(＞0.1μm)、SPP-2(＜0.1μm且＞50 KDa)和SPP-3(＜50 KDa)。
研究了马尾藻料液浓度对膜分离的影响,在料液比分别为1:20(m/V)、1:30、1:100、1:150,压力为0.5 MPa,温度为20℃时,将脱蛋白后的提取液进行膜分离,结果表明随着料液比的增加,膜分离后的多糖损失越少。综合考虑,在膜分离前将待处理的溶液按1:150稀释。在温度20℃时,压力分别在0.1、0.3、0.5 MPa下进行膜分离,结果表明操作压力对多糖损失并无显著影响,在膜分离设备允许压力范围内,0.1-0.5 MPa都是适合的。
2马尾藻多糖的纯化
多糖组分SPP-1、SPP-2和SPP-3占粗多糖百分比分别为72.15%、16.69%和11.16%,其中的多糖含量依次为58.13%、18.98%和16.38%,蛋白含量依次为1.09%、1.75%和1.73%,硫酸基含量依次为15.68%、15.26%和22.62%,糖醛酸含量依次为14.22%、14.30%和5.20%。
利用DEAE Cellulose-52层析柱分别对SPP-1、SPP-2和SPP-3三个多糖组分进行分离纯化,其中由SPP-1得到两个主要的组分SPP-1-1和SPP-1-2,得率分别为32.53%和8.67%;由SPP-2得到SPP-2-1、SPP-2-2和SPP-2-3三个组分,得率分别为10.53%、9.20%和36.33%;由SPP-3得到SPP-3-1和SPP-3-2两个组分,得率分别19.52%和57.27%。在纯化过程中多糖有部分损失,同时粗多糖中的蛋白基本被除去。利用Sephadex G-100层析柱分别对SPP-1-1、SPP-1-2、SPP-2-1、SPP-2-2、SPP-2-3、SPP-3-1和SPP-3-2七个组分进行分离纯化,以用于进一步的结构分析。
3马尾藻多糖的体外抗肿瘤活性与抗氧化活性
以MTT比色法探讨了经DEAE Cellulose-52纯化得到的七个多糖组分对人肺癌细胞株A549、人肝癌细胞株HepG2和人胃癌细胞株MGC-803生长的体外抑制作用。组分SPP-3-1和SPP-3-2对人肝癌细胞株HepG2、人肺癌细胞株A549和人胃癌细胞株MGC-803的生长具有明显的体外抑制作用,且抑制能力具有剂量依赖性。组分SPP-2-3只对肝癌HepG2细胞表现出强抑制率,并呈剂量依赖性。组分SPP-3-1和SPP-3-2具有较高的体外抗肿瘤活性可能与它们的低分子量(＜50 KDa)及高硫酸基含量(22.62%)有关。
在浓度为2 mg/mL时,粗多糖组分SPP-1、SPP-2和SPP-3对DPPH自由基的清除率分别为23.84%、13.23%和12.83%;总抗氧化能力分别为0.087、0.083和0.088(μmolFeSO_4/mg);还原力(以700 nm处的吸光度A_(700)表示)分别为0.785、0.446和0.541;对·OH的清除率分别为68.39%、13.22%和27.59%。
研究了乙醇浸提温度对马尾藻中总多酚含量的影响,在30℃马尾藻中的多酚类物质不能完全浸提出来;在70℃因浸出时间短不易控制,所以选择50℃作为乙醇浸提温度。马尾藻经50℃、70%乙醇浸提后,依次用不同有机溶剂萃取后得到乙酸乙酯萃取物、正丁醇萃取物及萃取剩余物,其占总萃取物百分比依次为15.76%、5.28%、78.96%,采用Folin-Ciocalteu比色法测定其中的总多酚含量,以绿原酸(CHA)作为标准物质,依次为3.20 mgCHA/g干重、12.12 mgCHA/g干重和52.08 mgCHA/g干重,总萃取物得率为8.07%。
乙酸乙酯萃取物和正丁醇萃取物在浓度为2 mg/mL时对DPPH自由基清除率分别为30.59%和29.36%。乙酸乙酯萃取物和正丁醇萃取物的总抗氧化能力较高,分别为0.520和0.345(μmolFeSO_4/mg)。乙酸乙酯萃取物、正丁醇萃取物和萃取剩余物的还原力较高,由高到低为萃取剩余物＞正丁醇萃取物＞乙酸乙酯萃取物。乙酸乙酯萃取物、正丁醇萃取物和萃取剩余物在浓度为2 mg/mL时对金属离子的螯合率分别为48.25%、51.34%和17.21%。
4马尾藻活性多糖的结构分析
利用高效凝胶过滤色谱法(HPGFC)测定多糖组分SPP-2-3、SPP-3-1、SPP-3-2的分子量和检测其纯度,其平均分子量(M_w)依次为132.999 KDa、5.874 KDa、7.249KDa。气相色谱法分析多糖的单糖组成,SPP-2-3单糖组成及摩尔比为鼠李糖:木糖:岩藻糖:甘露糖:葡萄糖:半乳糖=0.15:2.74:0.64:1.00:0.49:1.11,SPP-3-1单糖组成及摩尔比为甘露糖:葡萄糖:半乳糖=1.00:11.18:0.96,SPP-3-2单糖组成及摩尔比为鼠李糖:木糖:岩藻糖:甘露糖:葡萄糖:半乳糖=0.17:2.53:0.61:1.00:0.46:0.92。通过红外吸收光谱(IR)分析得知SPP-2-3和SPP-3-2中存在(?)糖苷键。
高碘酸氧化、Smith降解以及甲基化分析结果显示SPP-2-3中的甘露糖的连接方式以(1→3)糖苷键为主,葡萄糖和半乳糖除了存在(1→3)键型外,还存在(1→6)键连接方式;木糖的连接方式以(1→2)键为主;岩藻糖除了存在(1→3)键型外,还存在(1→4)键型。SPP-3-2中的甘露糖和半乳糖的连接方式以(1→3)键为主;葡萄糖是以(1→6)或者(1→2)键型连接;木糖和岩藻糖以(1→2)或(1→4)键型连接为主。
Sargassum pallidum belongs to Phaeophyta Sargassaceae and are extensively distributed in China Yellow Sea and Bohai Sea.Due to not being edible,S.pallidum is used in relatively narrow scope at present,and only a small part is used as feed,algin and raw materials of pharmaceutical industry,and most of them has not yet been fully utilized.Alga glycine is a kind of miscellaneous polysaccharide.It has a variety of physiological functions,and is an important bioactive substance in brown alga.However,little information about S.pallidum polysaccharides(SPP) and biological activity is available compared with those of the other Sargassum genus at present.In this paper S.pallidum collected from Rongcheng in Shandong Province was studied,including the antitumor activity and antioxidant activity in vitro of SPP,and identification,of polysaccharides structure.
This paper is composed of four parts:extraction of polysaccharides from S.pallidum, purification and identification of physical-chemical characters,antitumor activity and antioxidant activity of SPP in vitro,and identification of polysaccharides structure.Main results are listed as follows:
1.Extraction of S.pallidum polysaccharides
Supercritical CO_2(SC-CO_2) extraction was used to degrease the lipophylic substances in S.Pallidum.The effect of extraction pressure,extraction temperature and extraction time were investigated.As results,the optimum operating conditions for treatment were as follows:temperature at 55℃,pressure at 45 MPa,a flow rate of CO_2 at 20 L/h,and extraction time for 6 h,and the yield was 8.53‰.
Extraction of S.pallidum polysaccharide was optimized by the method of reSPPonse surface analysis(RSA).The effects of the main variables namely:ultrasonic time,extraction temperature and extraction time as well as the mutual interaction between variables were studied.The maximum yield of polysaccharides was up to 2.48%under the conditions as follows:ultrasonic time 469.54 s;extraction temperature 90.94℃;extraction time 4.96 h. The conditions of deproteinization by trichloroaeetie acid(TCA) were optimized.TCA final concentration was about 2%,and precipitation time was 2 h or longer.
Membrane separation was performed at 0.5 MPa and at 20℃.The deproteinized polysaccharides solution was pumped to membrane surface(tangential flow) with different nominal molecular weight cut-offs(0.1μm,100 KDa,50 KDa,10 KDa,3 KDa),in turn. The results showed that S.pallidum polysaccharides were mainly included in the fractions with molecular weight more than 50 KDa.Due to the fraction with pore size more than 0.1μm contained much polysaccharides,having complex composition,it was collected and analyzed singly.Fractions with molecular weight less than 50 KDa containing less polysaccharide were combined.In later experiments S.pallidum polysaccharides(SPP) were divided into 3 main crude polysaccharides,SPP-1(＞0.1μm),SPP-2(＜0.1μm and＞50 KDa) and SPP-3(＜50 KDa).
Effect of the ratio(material to water) on membrane separation was studied.At the ratio(material to water) of 1:20(m/V),1:30,1:100 and 1:150,pressure at 0.5 MPa, temperature at 20℃,the deproteinized polysaccharides solution were treated by membrane separation.The results showed that a higher ratio(material to water) led to a less loss of polysaccharides in membrane separation.It was considered to be suitable to dilute the solution at the ratio of 1:150 before membrane separation.At 20℃,membrane separation was performed at the pressure of 0.1,0.3 and 0.5 MPa,respectively.The results showed that the operating pressure had no significant effect on polysaccharides loss.So the pressure between 0.1 and 0.5 MPa are suitable in the scope allowed by membrane separation equipment.
2.Purification of S.pallidum polysaccharides
The yield of SPP-1,SPP-2 and SPP-3 accounted for 72.15%,16.69%and 11.16%, respectively of total crude polysaccharides.The polysaccharides contents were 58.13%, 18.98%and 16.38%,respectively and the protein contents were 1.09%,1.75%and 1.73%, respectively.The sulfate contents were 15.68%,15.26%and 22.62%,respectively and the uronic acid contents were 14.22%,14.30%and 5.20%,respectively.
A lyophilized fraction of polysaccharides SPP-1 was chromatographed on a DEAE Cellulose-52 anion-exchange column to yield two peaks,SPP-1-1 and SPP-1-2.In a similar manner,SPP-2-1,SPP-2-2 and SPP-2-3,SPP-3-1 and SPP-3-2 were obtained from SPP-2 and SPP-3,respectively.The recovery of SPP-1-1 and SPP-1-2 were 32.53%and 8.67%, respectively.The recovery of SPP-2-1,SPP-2-2 and SPP-2-3 were 10.53%,9.20%and 36.33%,respectively.The recovery of SPP-3-1 and SPP-3-2 were 19.52%and 57.27%, respectively.During the purification of crude polysaccharides a little part of polysaccharides were losed,while the protein in crude polysaccharide was almost removed. Fractions of polysaccharides SPP-1-1,SPP-1-2,SPP-2-1,SPP-2-2,SPP-2-3,SPP-3-1 and SPP-3-2,respectively were chromatographed on a Sephadex G-100 chromatography column.So they could be used for further structural analysis.
3.Antitumor activity and antioxidant activity in vitro of S.pallidum polysaccharides
The inhibition effects of 7 fractions purified by DEAE Cellulose-52 on the growth of HepG2 cells,A549 cells and MGC-803 cells were evaluated in vitro by MTT assay. Fractions of polysaccharides SPP-3-1 and SPP-3-2 presented significantly higher antitumor activity against the HepG2 cells,A549 cells and MGC-803 cells in vitro than blank control groups,and the inhibition ability was dose-dependent.SPP-2-3 only presented significantly higher antitumor activity against the HepG2 cells in vitro than a blank control, dose-dependently.It seems that SPP-3-1 and SPP-3-2 from SPP-3 with relatively lower molecular weight(＜50KDa) and higher sulfate contents(22.62%) showed significantly higher antitumor activity against the HepG2 cells,A549 cells and MGC-803 cells in vitro.
At the concentration of 2mg/mL,scavenging of DPPH radical of crude polysaccharide SPP-1 and SPP-2 and SPP-3 were 23.838%,13.232%and 12.828%,respectively.The total antioxidant capacity of SPP-1 and SPP-2 and SPP-3 were 0.087,0.083 and 0.088, respectively(μmolFeSO4/mg).The reducing power(expressed as absorbanee at 700 nm) and OH radical scavenging of SPP-1,SPP-2 and SPP-3 increased with the increasing concentration in experimental scope.Their reducing power were 0.785,0.446 and 0.541, respectively at 2 mg/mL.Their OH radical scavenging were 68.391%,13.218%and 27.586%,respectively at 2 mg/mL.
Effect of ethanol extraction temperature on total polyphenol content extracted from S. pallidum were studied.At 30℃total polyphenol could not been fully extracted from S. pallidum,while at 70℃the extraction time was too short to be controlled,so 50℃was selected as alcohol extraction temperature.S.pallidum was extracted by 70%ethanol at 50℃,and then extracted by chloroform,ethyl acetate and n-butanol in turn.The yeied of total extract was 8.07%.Ethyl acetate extract,n-butanol extract and extract residue accounted for 15.76%,5.28%and 78.96%,respectively of total extract,and their total polyphenol contents were 3.20 mgCHA /gDW,12.12 mgCHA/gDW and 52.08 mgCHA/gDW, respectively determined by Folin-Ciocalteu method.
DPPH radical scavenging of ethyl acetate and n-butanol extract were 30.59%and 29.36%,respectively at 2 mg/mL.Their total antioxidant capacity were 0.520 and 0.345 (μmolFeSO4/mg),respectively.Reducing power of ethyl acetate extracts,n-butanol extract and extract residue were high.The descending sequence was extract residue,n-butanol extract and ethyl acetate extracts.Metal ions scavenging of ethyl acetate extracts,n-butanol extracts and extract residue were 48.25%,51.34%and 17.21%at 2 mg/mL.
4.Structural analysis of S.pallidum polysaccharides
The average molecular weight and purity of polysaccharides fractions SPP-2-3, SPP-3-1 and SPP-3-2 were defined by high performance of gel filtration chromatography (HPGFC).They were 132.999KDa,5.874KDa and 7.249KDa,respectively. Monosaccharide composition and molar ratio of SPP-2-3,SPP-3-1 and SPP-3-2 were analyzed by Gas chromatography(GC).They were rhamnose:xylose:fucose:mannose: glucose:galactose= 0.15:2.74:0.64:1.00:0.49:1.11,mannose:glucose:galactose= 1.00:11.18:0.96,and rhamnose:xylose:fucose:mannose:glucose:galactose=0.17: 2.53:0.61:1.00:0.46:0.92,respectively.The infrared absorption spectroscopy(IR) of SPP-2-3 and SPP-3-2 showed a character of口-glycosidic bond.
Periodate oxidation,Smith degradation and methylation analysis of SPP-2-3 showed that mannose were linked mainly by(1→3) glycosidic bond.In addition to(1→3) glycosidic bond in glucose and galactose,there was also(1→6) glycosidic bond.Xylose was linked mainly by(1→2) glycosidic bond.In addition to(1→3) glycosidic bond in fucose,there was also(1→4) glycosidic bond.For SPP-3-2,mannose and galactose were linked mainly by(1→3) glycosidic bond.Glucose was linked by(1→6) or(1→2) glycosidic bond.Fucose and L-xylose was linked by(1→2) or(1→4) glycosidic bond.
引文
[1]吕惠敏,张侃.海藻的利用与开发[J].食品科技,1998,6:29-30
[2]李来好,杨贤庆,吴燕燕.马尾藻的营养成分分析和营养学评价[J].青岛海洋大学学报,1997,27(3):319-325
[3]李刘冬,李来好,石红,等.海藻膳食纤维对雌激素吸附作用的研究[J].中国海洋药物,2005,24(3):1-4
[4]陈培基,李刘冬,李来好,等.二种褐藻膳食纤维的功能活化及其生理功能的比较[J].食品科学,2005,26(1):226-230
[5]李刘冬,李来好,陈培基,等.马尾藻袋泡茶的生产工艺与条件[J].食品与发酵工业,2003,29(9):31-34
[6]李来好,吴燕燕.马尾藻袋泡茶的生产技术[J].广州食品工业科技,1995,11(2):50-51
[7]丁恒山.中国药用孢子植物[M].上海:科学技术出版社,1982
[8]曾呈奎,陆保仁.中国海藻志[M].第三卷,第二册.北京:科学出版社,2001
[9]中华人民共和国卫生部药典委员会编.中华人民共和国卫生部药典[M].一部.北京:人民卫生出版社,1990
[10]陈锡林.浙江海洋药用藻类资源调查研究初报[J].浙江中医学院学报,2000,24(2):65-67
[11]崔征,李玉山,肇文荣.中药海藻及数种同属植物的药理作用[J].中国海洋药物,1997,(3):5-8
[12]刘秋英,孟庆勇.半叶马尾藻多糖体内抗肿瘤作用及其机制探讨[J].第一军医大学学报,2004,24(4):434-436
[13]孟庆勇,刘志辉,郑辉.半叶马尾藻多糖对辐射损伤小鼠骨髓细胞集落生成的影响[J].青岛海洋大学学报,1997,35(3):503-506
[14]Na H J,Moon P D,Ko S G,et al.Sargassum hemiphyllum inhibits a topic reaction via the regulation of inflammatory mediators[J].J Pharmacol Sci,2005,97(2):19-26
[15]严小军.马尾藻碘化学形态研究突破(南方产马尾藻碘含量最高)[J].海洋科学,2000,24(3):56
[16]韩丽君,范晓,周天成,等.广西北部湾马尾藻脂肪酸组成的研究[J].海洋科学集刊,1995,36:175-180
[17]蔡双莲,李敏.多不饱和脂肪酸的研究进展[J].生命科学研究,2003,7(4):289-304
[18]Yeh S T,Lee C S,Chen J C.Administration of hot-water extract of brown seaweed Sargassum duphcatun via immersion and injection enhances the immune resistance of white shrimp Litopenaeus vannamei[J].Fish Shellfish Immunol,2006,20(3):32-45
[19]常巧玲,孙建义.海藻饲料资源及其在水产养殖中的应用研究[J].饲料工业,2006,27(2):62-64
[20]王如才,于瑞海.海藻榨取液在海湾扇贝亲贝蓄养中的应用[J].海洋科学,1989,6:55-56
[21]潘鲁青,张涛.海藻磨碎液饲育中国对虾幼体试验[J].海洋科学,1997,2:3-5
[22]Chotigeata W,Tongsupa S,Supamataya K,et al.Effect of Fucoidan on Disease Resistance of Black Tiger Shrimp[J].Aquaculture,2004,233(1-4):23-30
[23]周歧存,赵华超.海藻在罗氏沼虾饲料中的应用研究[J].饲料研究,2001(8):5-8
[24]周歧存,肖风波.海藻在南美白对虾饲料中的应用研究[J].海洋科学,2003,27(3):66-69
[25]Bindu M S,Sobha V.Conversion efficiency and nutrient digestibility of certain seaweed diets by laboratory reared Labeorohita(Hamilton)[J].Indian J Exp Biol.2004,42(12):39-44
[26]赵学武,徐鹤林.关于马尾藻代替部分粮食和矿物质饲喂肉鸡的探讨[J].青岛海洋大学学报,1990,20(1):86-91
[27]张善明,刘强,张善垒.从海带中提取高粘度海藻酸钠[J].食品工业科技,2002,(3):72-73
[28]侯振建,刘婉乔.从马尾藻中提取高粘度海藻酸钠[J].海洋科学,1997,(5):18-20
[29]邹荣岳.从马尾藻中生产海藻酸钠[J].广西科学学院学报,1999,(2):81-83
[30]何培民,徐姗楠,张寒野.海藻在海洋生态修复和海水综合养殖中的应用研究简况[J].渔业现代化,2005(4):15-16
[31]Tsui M T,Cheung K C,Tam N F.A comparative study on metal sorption by brown seaweed[J].Chemosphere.,2006,65(1):1-7
[32]杨宇峰,宋金明,林小涛.大型海藻栽培及其在近海环境的生态作用[J].海洋环境科学,2005,24(2):77-80
[33]Terawali T,Yoshikawa K,Yoshida G.Ecology and restoration techniques for Sargassum beds in the Seto Inland Sea,Japan[J].Mar Pollut Bull,2003,47:198-201
[34]Crouch I J,Beckett R P,Van Stadan.J.Effects of seaweed concentrate on the growth and mineral nutrient stressed lettuce[J].J.Applied Phycology,1990,2:269-272
[35]汤学军,潘列梅,刘慧玲.马尾藻活性物质对水稻幼苗抗旱性的作用[J].海洋科学,2004(10):45-47
[36]Sivasankari S,Venkatesalu V,Anantharaj M.Effect of seaweed extracts on the growth and biochemical constituents of Vigan sinensis[J].Bioresour Technol.2006,97(14):45-51
[37]王平.褐藻胶在日用化学品中的应用[J].现代日用科学,2002,(5):44
[38]张惟杰主编.糖复合物生化研究技术[M].浙江大学出版社,第二版,2003
[39]庞启深,郭宝江,阮继红.螺旋藻多糖对核酸内切酶活性和DNA修复合成的增强作用[J].遗传学报,1988,15:374-381
[40]周慧萍,蒋巡天,陈琼华等.浒苔多糖的降血脂及其对SOD活力和LPO含量的影响[J].生物化学杂志,1995(11):161-165
[41]徐旭,于冰,汤立达,等.海洋生物多糖的药用功能[J].天津药学,2004,16(6):51-53
[42]薛静波,刘希英,张鸿芬.海带多糖对小鼠腹腔巨噬细胞的激活作用[J].中国海洋药物,1999,18(3):23-25
[43]Okai Y,Ishizaka S,Higashi-Okai K,et al.Detection of immunomodulating activities in an extract of Japanese edible seaweed,Laminaria japonica[J].J Sci Food Agric,1996,72(4):455-460
[44]Zvyagintseva,Tatiana N.Inhibition of complement activation by water-soluble polysaccharides of some fareastern brown seaweeds[J].Comp Biochem Physiol,Part C:Toxicol Pharmacol,2000,126C(3):209-215
[45]苗本春,耿美玉,李静,等.海洋硫酸多糖911免疫增强作用的探讨[J].中国海洋药物,2002,21(5):1-4
[46]黄益丽,郑忠辉,苏文金等.二色桌片参的化学成分研究:Ⅲ.二色桌片参多糖-1-岩藻聚糖的免疫调节[J].海洋通报,2001,20(1):88-91
[47]陈文星,吴皓,金亦涛.珠蚌多糖的免疫调节作用研究[J].中药药理与临床,2001,17(5):16-18
[48]朱爱民,扬晓峰,李晟刚,等.螺旋多糖对小鼠免疫系统的作用[J].山东医药工业,2001,20(6):44-45
[49]陈绍瑗,莫卫民,潘远江.海洋药物研究(Ⅲ)——羊栖菜多糖[J].兰州大学学报(自然科学版),1998,34(4):110-113
[50]徐明芳,高孔荣,刘婉乔.海藻保健功能的研究进展[J].食品科学,1995,16(12):19-22
[51]汪开治.海藻含有免疫抑制物[J].生物学通报,1995,30(1):11
[52]姚福汉,蒋伯诚.海藻中具有免疫赋活作用的多糖[J].中国野生植物资源,1997,16(3):6-7
[53]王庭欣,秦淑贞,赵文,等.海藻多糖对环磷酰胺诱发小鼠骨髓微核率的抑制作用[J].癌变·畸变·突变,1999,11(2):106-107
[54]王庭欣,蒋东升,赵文,等.海带多糖对小鼠免疫功能的影响[J].中国老年学杂志,1999,19(专刊):12
[55]徐惠,于志洁,等.螺旋藻黏多糖的分离及其免疫学作用[J],中国生化药物杂志,1997,18(2):72-74
[56]Li J Z,Bao C X,Chen G Z.Antithrombin activity and platelet aggregation by acid mucopolysaccharides isolated from stichopus japonicus selenka.China[J].Acta Pharmacologica Sinica,1985,6(2):107-110
[57]马西,Lindhout T,Beguin S,et al.刺参酸性粘多糖抑制凝血酶活性的辅因子[J].中华血液学杂志,1990,11(5):241-243
[58]Nagase H,Enjyoji K I,Minamiguchi K,et al.Depolymerized holothurian glycosaminoglycan with novel anticoagulant actions:antithrombin Ⅲ-and heparin cofactor Ⅱ-independent inhibition of factor X activation by factor LXa-factor Ⅷa complex and heparin cofactorⅡ-dependent inhibition of thrombin[J].Blood,1995,85:1527-1534
[59]王学锋,李志广,储海燕,等.海参糖胺聚糖抗血栓形成机制的研究[J].中国新药与临床杂志,2002,21(12):718-721
[60]刘志峰,宫晓黎,魏淑贞.五种海藻多糖体外抗血小板聚集作用观察[J].中国海洋药物,2001,20(2):36-38
[61]辛现良,耿美王,戚欣,等.新型抗脑缺血海洋新药989作用机理的初步探讨[J].青岛海洋大学学报,2001,31(4):536-540
[62]耿美玉,辛现良,李静,等.新型海洋硫酸多糖DPS对大鼠脑缺血的保护作用[J].海洋与湖沼,2001,32(2):192-197
[63]刘秋英,孟庆勇,刘志辉.海藻多糖抗肿瘤作用的研究进展[J].中国海洋药物,2003,22(4):45-48
[64]曲显俊,崔淑香,解砚英.螺旋藻多糖抗癌作用的实验研究[J].中国海洋药物,2000,19(4):10-14
[65]许东晖,许实波,王兵等.皱纹盘鲍多糖抗肿瘤药理作用研究[J].热带海洋,1999,18(4):86-90
[66]Ellouali M,Boisson V C,Durand P,et al.Antitumor activity of low molecular weight fucans extracted from brown seaweed Ascophyllum nodosum[J].Anticancer Res,1993,13:2011-2020
[67]Mishima T,Murata J,Toyoshima M,et al.Inhibition of tumor invasion and metastasis by calcium spirulan(Ca-SP),a novel sulfated polysaccharide derived from a blue-green alga,Spirulina platensis[J].Clinical and Experimental Metastasis,1998,16(6):541-550
[68]卢睿春,侯振建,刘婉娇.亨氏马尾藻硫酸多糖抗肿瘤活性的研究[J].海洋科学,1998,3:63-64
[69]曾波航,王光明,曾亚仑.钝顶螺旋藻多糖对急性白血病病人NK细胞的作用[J].中国海洋药物,2000,(6):45-47
[70]寿佩勤.部分海藻多糖的抗肿瘤作用研究和临床应用[J].现代实用医学,2004,16(7):432-434
[71]陈毓强,吴炳南.四种多糖化合物的抗癌作用及其对免疫功能的影响[J].癌症,1997,16(3):198-200
[72]张朝晖,蔡宝昌.海洋药物研究与开发[M].北京:人民卫生出版社,2003
[73]耿美玉,辛现良,管华诗.海洋硫酸多糖类药物911抗艾滋病毒作用及其机理研究[J].中国药理学会通讯,2001,18(4):13-23
[74]于红,张学成.螺旋藻多糖抗HSV-1作用的体外实验研究[J].高技术通讯,2002,12(9):65-69
[75]Albertus J S.Medicinal and pharmaceutical uses of seaweed natural products:A review[J].Journal of Applied Phycology,2004,16:245-262
[76]魏文青,王明霞,丛建波,等.海藻硫酸多糖抗乙型肝炎病毒的实验研究[J].中华肝脏病杂志,2002,10(2):112
[77]王秉及.海洋药物的抗病毒研究[J].生物工程进展,1996,16(6):34-36
[78]王安利,胡俊荣.海藻多糖生物活性研究进展[J].海洋科学,2002,26(9):36-39
[79]徐明芳,高孔荣,刘婉乔.海藻保健功能的研究进展[J].食品科学,1995,16(12):19-22
[80]王炳岩,季宇彬.海藻多糖对白血病L615小鼠LPO含量及GR、GSH—PX、CAT、SOD酶活性的影响[J].中医药信息,1994,11(5):43-45
[81]李妍,田晓华,丛建波,等.海藻多糖抑制白细胞呼吸爆发作用研究[J].生物化学与生物物理进展,1999,26(2):162-164
[82]刘凤艳,钟红茂,范洁伟,等.海藻多糖药理作用研究新进展[J].广东药学,2005,15(3):81-84
[83]Kumar G P,Suheesh S,Vijayalakshmi N R.Hypoglyeemic effect of coecinia indica,mechanism of action[J].Planta Med,1993,59(4):330-332
[84]侯建明,蓝进.海洋生物多糖(AC-1)防治高脂血症的实验报告[J].海军医学杂志,2001,22(3):197-199
[85]王远红,徐家敏,耿美玉,等.海洋硫酸多糖916对实验性高脂血症大鼠血浆中含硫氨基酸的影响[J].中国海洋药物,2000,19(4):23-24
[86]王兵,李靖,马舒冰,等.羊栖菜多糖降血糖的实验研究[J],中国海洋药物,2000,3:33-35
[87]李八方,毛文君,曹立民.羊栖菜多糖对高血脂模型大鼠血脂的影响[J].中国水产科学,2000,7(2):56-58
[88]周慧萍,陈琼华.紫菜多糖的抗凝血和降血脂作用[J].中国药科大学学报,1990,21(6):358-360
[89]邓槐春,谈竞光,谢娇娥等.海藻多糖对高血脂症的临床疗效初步观察[J].中草药,1987,18(8):12
[90]阮积惠.海藻主要药用成分的研究和展望[J].东海海洋,2001,19(2):1-9
[91]范晓,周永航.海藻中的甜菜碱及其类似物[J].海洋科学,1999,23(3):24-26
[92]张成武,曾昭琪,张嫒贞,等.钝顶螺旋藻多糖和藻蓝蛋白对小鼠急性放射病的防护作用[J].营养学报,1996,3:327-331
[93]李德远,王海滨,张声华,等.海带岩藻糖胶及褐藻胶抗辐射效应研究[J].武汉食品工业学院学报,1999,2:18-22
[94]王友顺,黎露刚,吴侃,等.螺旋藻多糖对给予环磷酰胺BALB/C小鼠的保护作用[J].海洋科学,1997,6:36-38
[95]王友顺,黎露刚,吴侃,等.螺旋藻多糖对CD3AK细胞增殖能力的影响[J].海洋科学,1998,3:37-39
[96]王庭欣,秦淑贞,赵文,等.海带多糖对环磷酰胺诱发小鼠骨髓细胞微核率的抑制作用[J].癌变.畸变.突变,1999,11(2):106-107
[97]田晓华,丛建波,施定基,等.褐藻多糖硫酸酯清除活性氧自由基作用及动力学的ESR研究[J].营养学报,1997,19(1):32-37
[98]藤霞,丛建波,田晓华,等.海藻硫酸多糖抗氧化与抗肿瘤作用的实验研究[J].营养研究,1998,20(1):48
[99]詹林盛,张新生,吴小红,等.褐藻多糖对氧化应激诱导的淋巴细胞DNA损伤的保护作用[J].中国海洋药物,1999,4:4-7
[100]石达友,刘汉儒,黎建华.中药对鸡新城疫灭活疫苗免疫效果的影响[J].中国兽医科技,2004,34(2):42-46
[101]李子昆.海藻提物的减肥及保湿作用研究[J].四川日化,1997,(1):12-15
[102]诸葛健,赵振锋,方慧英.功能性多糖的构效关系[J].无锡轻工大学学报,2002,21:209-212
[103]Ishibashi K,Miura N N,Adachi Y,et al.Relationship between solubility of grifolan,a fungall,3-β-D-glucan,and production of tumor necrosis factor by macrophages in vitro[J].Biosci Biotechnol Biochem,2001,65(9):1993-2000
[104]田庚元,冯宁澄.多糖类免疫调节剂的研究和应用[J].化学进展,1994,6(2):114-124
[105]王长云,管华诗.多糖抗病毒研究进展Ⅱ:硫酸多糖抗病毒作用[J].生物工程进展,2000, 20:3-8
[106]Ehresmann D W,Deig E F,Hatch M T,et al.Dextran sulfate,a potent anti-HIV agent in vitro having synergism with sidovudine[J].Lancet,1987,1:1379-1386
[107]李艳辉,王琦.真菌多糖的生物活性与构效关系研究评价[J].吉林农业大学学报,2002,24(2):70-77
[108]王兆梅,李琳,郭祀远,等.活性多糖构效关系研究评述[J].现代化工,2002,22(8):18-22
[109]王超英,吕金顺,邓芹英.菌类多糖的生物活性与结构关系[J].天水师范学院学报,2001,21(5):18-21
[110]杜巍,李元瑞,袁静.食药用茵多糖生物活性与结构的关系[J].食用菌,2001,(2):3-5
[111]方唯硕,刘相.具有抗HIV活性的天然产物[J].国外医学:中医中药分册,1993,8(2):61-69
[112]Saito H,Yoshioka Y,Uehara N,et al.Relationship between conformation and biological response for(1→3)-β-D-glucans in the activation of coagulation Factor G from limulus amebocyte lysate and host-mediated antitumor activity.Demonstration of single-helix conformation as a stimulant [J].Carbohydrate Research.,1991,217:181-190
[113]周庆华.超临界萃取技术最佳方法的研究[J].哈尔滨理工大学学报,2003,8(3):125-126
[114]何江川,韩永萍.超滤膜分离法在多糖分离提取中的应用[J].食用菌,2005,1:5-7
[115]赵晓燕,王长云.海洋多糖分子修饰方法研究概况[J].海洋科学,2000,24:20-22
[116]Sakai T,Kimura H,Kojima K,et al.Marine bacterial sulfated fucoglucuronomannan(SFGM)lyase digests brown algal SFGM into trisaccharides[J].Mar Biotechnol(NY),2003,5:70-78
[117]Miller I J,Blunt J W.Desulfation of algal galactan[J].Carbohydrate Research,1998,309:39-43
[118]梁忠岩,苗春艳,张翼伸,等.化学修饰对斜顶菌多糖抑瘤活性影响的研究[J].中国药学杂志,1996,30:613-615
[119]Uryu T,Ikushima N,Katsuraya K,et al.Sulfated alkyl oligoaacharides with potent inhibitory effects on HIV infection[J].Biochem Pharmacol,1992,43:2385-2392
[1]卢群,丘泰球,胡爱军.超声强化法提取海藻油的研究[J].广东药学院学报,2003,19(2):104-105
[2]张秀妍,姜爱莉,王长海.玻璃海鞘脂肪酸提取工艺的研究[J].海洋通报,2004,23(4):93-96
[3]Jamieson G R,Reid E H.The component acids of some marine algal lipids[J].Phytochemistry,1972,11:1423-1432
[4]刘志伟.超临界流体萃取技术及其在食品工业中的应用[J].食品研究与开发,2004,25(2):3-6
[5]刘雄,阚建全.超临界二氧化碳萃取和精制柚皮香精油的研究[J].食品与机械,2003,(4):13-14
[6]周庆华.超临界萃取技术最佳方法的研究[J].哈尔滨理工大学学报,2003,8(3):125-126
[7]颜英.超临界CO_2萃取沙棘油的研究[J].精细石油化工,2003,(3):39-41
[8]Annadurai G.Design of optimum reSPPonse surface experiments for adsorption of direct dye on Ehitosan[J].Bioproc Eng,2000,23:451-455
[9]Reddy P R M,Reddy G,Seenayya G.Produetion of the nnostable pullulanase by Clostridium thermosulfurogenes SV2 in solid-state fermentation:optimization of nutrients levels using reSPPonse surface methodology[J].Bioproc Eng,1999,21:497-503
[10]Rastogi N K,Rashmi K R.Optimisation of enzymatic liquefaction of mango pulp by reSPPonse surface methodology[J].Eur Food Res Technol,1999,209:57-62
[11]Gouveia E R,Baptista-Neto A.Optimisation of medium composition for clavulanic acid production by streptomyces clavuligerus[J].Biotechnol Lett,2001,23:157-161
[12]Ambati P,Ayyanna C.Optimizing medium constituents and fermentation conditions for citric acid production from Palmyra jaggery using reSPPonse surface method[J].World J Microbiol Biolotechnol,2001,17:331-335
[13]Chen Q H,He G Q,Mokhtar A M A.Optimization of medium composition for the production of elastase by Bacillus SPP.EL31410 with reSPPonse surface methodology[J].Enzyme Microbial Technol,2002,30:667-672
[14]Bradford M M.A rapid and sensitive method for the quantitation of microgram quantitatives of protein utilizing the principle of protein-dye binding[J].Anal Biochem,1976,72:248-254
[15]冀颐之,杜连祥.深层培养裂褶菌胞外多糖的提取及结构研究[J].微生物学通报,2003,30(5):15-20
[16]高居易,卓建亭,黄一红.紫孢侧耳多糖的提取、纯化及性质的研究[J].福建师范大学学报(自然科学版),1999,15(2):83-87
[17]何江川,韩永萍.超滤膜分离法在多糖分离提取中的应用[J].食用菌,2005,1:5-7
[18]张宁,彭志英.中空纤维超滤膜浓缩胞外多糖PS-9415发酵液的研究[J].Science and Technology of Food Industry,2003,24(2):24-27
[19]刘茉娥等编.膜分离技术[M].化工出版社:1998年第1版
[20]Dubois M,Crilles KA,Hamilton J K,et al.Colorimetric method for determination of sugars and related substances.Analytical Chemistry,1956,28:350-356
[21]陈均辉,陶力,李俊,等.生物化学实验[M].北京:科学出版社,2003
[1]Yamaguchi F,Ota Y,Hatanaka C.Extraction and purification of pectic polysaccharides from soybean okara and enzymatic analysis of their structures[J].Carbohydrate Polymers,1996,30:265-273
[2]Yu R M,Wang L,Zhang H,et al.Isolation,purification and identification of polysaccharides from cultured Cordyceps militaris.Fitoterapia,2004,75:662-666
[3]赵永芳.生物化学技术原理及应用[M].北京:科学出版社,2002
[4]方积年.多糖的分离纯化及其纯度鉴别与分子量测定[J].药学通报,2003,19(10):46-49
[5]李波,许时婴.羊栖菜褐藻糖胶的分级纯化和结构分析[J].无锡轻工大学学报,2004,23(2):86-89
[6]刘敦华,谷文英.沙蒿籽胶多糖的分级纯化和结构分析[J].食品科学,2007,28(3):73-76
[7]吴亚林,黄静,潘远江.无花果多糖的分离、纯化和鉴定[J].浙江大学学报:理学版,2004,31(2):177-179
[8]闻吉昌,催春月,张奕,等.芦荟多糖的分离纯化及结构分析[J].高等学校化学学报,2003,24(7):1189-1192
[9]张金亭,黄勇,李金话,等.小刺猴头多糖I的分离纯化及组成分析[J].中国医学杂志,2005,40(7):545-547
[10]Lloyd A G,Dodgson K S,Price RG,et al.Polysaccharide sulphates[J].Biochimica et Biophysica Acta,1961,1:108-115
[11]Bitter T,Muir H M.Amodified uornic acid carbazole reaction[J].Analytical Biochemistry 1962,(4):330-334
[1]Yoshiaki F,Youichi S,Ko-ichi O.Selective tumoricidal effect of soluble proteoglucan extracted from the basidiomycete,Agaricus blazei Murill,mediated via natural killer cell activation and apoptosis [J].Cancer Immunol Immunother,1998,46:147-159
[2]Yoshiaki F.Tumor-SPPecific cytocidal and immunopotentiating effects of relatively low molecular weight products derived from the Basidiomycete,Agaricus blazei Murill[J].Anticancer Res,1999,19:113-118
[3]毛俊好.白术多糖对小鼠淋巴细胞功能的调节[J].免疫学杂志,1996,12(4):233-236
[4]向道斌,李晓玉.牛膝多糖的抗肿瘤活性及其免疫增强作用(英文)[J].中国药理学报,1993,14(6):556-561
[5]Adachi Y,Okazaki M,Ohno N,et al.Enhancement of cytokine porduction by macrophages stimulated with 口(1口 3)-D-giucan,grifolan(CRN),isolated from Grifola frondosa[J].Biological and Pharmacentical Bulletin,1994,17(12):1554-1560
[6]韩锐主编.抗癌药物研究与实验技术[M].北京:北京医科大学协和医科大学联合版社,1997
[7]Ito H,Shimura K,Itoh H,et al.Antiutmor effects of a new polysaccharide-protein complex(ATOM)prepared from Agaricus blazei(Iwade srtain101)"Himematsutake" and its mechanisms in tumor-bearing mice[J].Anticancer Res,1997,17:277-284
[8]王葡文主编.实验肿瘤学基础[M].北京:人民卫生出版社,1992:279-300
[9]张尔贤,俞丽君.鼠尾藻多糖清除氧自由基作用的研究[J].中国海洋药物,1997,3:1-4
[10]于广利,吕志华,王曙光,等.海黍子提取物对不饱和脂质抗氧化作用[J].青岛海洋大学学报,2000,30(1):75-80
[11]于广利,薛长湖,楼伟风.海藻多糖类化合物对卵磷脂氧化的抑制作用[J].中国水产科学,1998,5(4):48-51
[12]师然新,徐祖洪.青岛沿海九种海藻的类脂及酚类抗菌活性的研究[J].中国海洋药物,1997,4:16-19
[13]Matsukawa R,Dubinsky Z,Kishimoto E,et al.A comparison of screening method for antioxidant activity in seaweeds[J].J Appl Phycol,1997,9:29-35
[14]Nakamura T,Nagayama K,Uchida K,et al.Antioxidant activity of phlorotannins isolated from the brown alga Eisenla bycyclis[J].Fisheries Science,1992,62(6):923-926
[15]石碧,狄莹著.植物多酚tM].北京:科学出版社,2000,124-134
[16]Yan X J,Li X C,Zhou C X,et al.Prevention of fish oil rancidity by phlorotannins from Sargassum kjellmanianum[J].J.Appl.Phycol.,1996,8:201-203
[17]Leong L P,Shui G.An investigation of antioxidant capacity of fruits in Singapore markets[J].Food Chemistry,2002,76(1):69-75
[18]Benzie I F,Strain J J.The ferric reducing ability of plasma(FRAP) as a measure of"antioxidant power":the FRAP assay[J].Journal of Analytical Biochemistry,1996,239(1):70-76
[19]Yen G C,Chen H Y.Antioxidant activity of various tea extracts in relation to their antimutagenicity.J Agric Food Chem,1995,43:27-32
[20]Wang J C,Xing G S,Hu W D,et al.Effects of Ge-132 on oxygen free radicals and the lipid peroxidation induced by hydroxyl free radical in vitro.Chinese Pharmaceutical Journal,1994,29:23-25
[21]Nurmi K,Ossipov V,Haukioja E,et al.Variation of total phenolic content and individual low-molecular-weight phenolics in foliage of mountain birch trees(Betula pubescens sSPP.tortuosa)[J].J Chem Ecol,1996,22:2023-2040
[22]Decker E A,Welch B.Role of ferritin as a lipid oxidation catalyst in muscle food.J Agric Food Chem,1990,38:674-677
[23]Dias P F,Siqueira J M,Vendrascolo L F,et al.Antiangiogenic and antitumoral properties of a polysaccharide isolated from the seaweed Sargassum stenophyllum[J].Cancer Chemotherapy and Pharmacology,2005,56(4):436-446
[24]Duarte M E R,Cardoso M A,Noseda M D,et al.Structural studies on fucoidans from the brown seaweed Sargasswn stenophyllum[J].Carbohydrate Research,2001,333(4):281-293
[25]王长云,管华诗.糖抗病毒作用研究进展Ⅱ硫酸多糖抗病毒作用[J].生物工程进展,2000,20(2):3-8
[26]Zhang M,Zhang L,Cheung P C K,et al.Molecular weight and anti-tumor activity of the water-soluble polysaccharides isolated by hot water and ultrasonic treatment from the sclerotia and mycelia of Pleurotus tuber-regium[J].Carbohydrate Polymers,2004,56:123-128
[27]Hu F L,Lu R L,Huang B,et al.Free radical scavenging activity of extracts prepared from fresh leaves of selected Chinese medicinal plants[J].Fitoterapia,2004,75(1):14-23
[28]Aruoma O I.Free radicals,oxidative stress,and antioxidants in human health and disease.J Am Oil Chem Soc,1998,75:199-212
[29]Shon M Y,Kim T H.Sung N J.Antioxidants and free radical scavenging activity of Phellinus baumii(Phellinus of Hymenochaetaceae) extracts.Food Chem,2003,82:593-597
[30]Jimenez-Escrig A,Jimenez-Jimenez I,Pulido R,et al.Antioxidant activity of fresh and processed edible seaweeds[J].Journal of the Science of Food and Agriculture,2001,81(5):530-534
[31]Yan X,Nagata T,Fan X.Antioxidative activities in some common seaweeds[J].Plant Foods for Human Nutrition,1998,520):253-262
[32]Xue C H,Fang Y,Lin H,et al.Chemical characters and antioxidative properties of sulfated polysaccharides from Laminaria japonica[J].Journal of Applied Phycology,2001,13(1):67-70
[33]Xue C,Yu G,Hirata T,et al.Antioxidative activities of several marine polysaccharides evaluated in a phoSPPhatidylcholine-liposomal suSPPension and organic solvents[J].Bioscicnce,Biotechnology,and Biochemistry,1998,62(2):206-209
[34]Ruperez P,Ahrazem O,Leal J A.Potential antioxidant capacity of sulfated polysaccharidcs from the edible marine brown seaweed Fucus vesiculosus[J].Journal of Agricultural and Food Chemistry,2002,50(4):840-845
[35]Yamaguchi F,Ariga T Yoshimira Y,et al.Antioxidant and anti-glycation of carcinol from Garcinia indica fruit rind.J Agric Food Chem,2000,48:180-185
[1]Miyazaki T,Nishijima M.Studies on fungal polysaccharides.ⅩⅩⅦ.Structural examination of a water-soluble,antitumor polysaccharide of Ganoderma lucidum[J].Chem Pharm Bull,1981,29:3611-3616
[2]Yamada H,Kawaguchi N,Ohmori T,et al.Structure and antitumor activity of an alkali-soluble polysaccharide from Cordyceps ophioglossoides[J].Carbohydr Res,1984,125:107-115
[3]Iino K,Ohno N,Suzuki I,et al.Structural characterization of a neutral antitumorβ-D-glucan extracted with hot sodium hydroxide from cultured fruit bodies of Grifola frondosa[J].Carbohydr Res,1985,141:111-119
[4]Misaki A,Nasu M,Sone Y,et al.Comparison of structure and antitumor activity of polysaccharides isolated from Fukurotake,the fruiting body of Volvariella volvacea[J].Agric Biol Chem,1986,50:2171-2184
[5]王森,丁霄霖.葡聚糖生物活性与结构的关系[J].无锡轻工大学学报,1997,(2):90-94
[6]Ning J,Zhang W H,Yi Y T,et al.Synthesis of β-(1-6)-ranched β-(1-3) glucohexanse and its analogues containing an α-(1-3) linked bond with antitumor activity[J].Bioorganic Med Chem,2003,11:2193-2203
[7]魏远安,方积年.高效凝胶渗透色谱法测定多糖纯度及分子量[J].药学学报,1989,24(7):532-536
[8]Hagel L.Comparison of some soft gels for the molecular-weight distribution analysis of dextran at enhanced flow-rates[J].J Chromatogr,1978,160:59-71
[9]董群,方积年.寡糖及多糖甲基化方法的发展及现状[J].天然产物研究与开发,1995,7(2):60-65
[10]徐桂芸,常理文.微量寡糖的甲基化分析-用于魔芋葡甘多糖酶解产物的结构测定[J].分析化学,1996,24(12):1400-1404
[11]Hakomori S.A rapid permethylation of glycolipid,and polysaccharide catalyzed by methylsulfinyl carbanino in dimethyl sulfoxide[J].J Biochem,1964,55:205-208
[12]Miyazaki T,Oikawa N,Yamada H,et al.Structural examination of antitumour,watersoluble glucans from Grifora umbellate by use of four types of glucanase[J].Carbohydr Res,1978,65:235-243
[2]李来好,杨贤庆,吴燕燕.马尾藻的营养成分分析和营养学评价[J].青岛海洋大学学报,1997,27(3):319-325
[3]李刘冬,李来好,石红,等.海藻膳食纤维对雌激素吸附作用的研究[J].中国海洋药物,2005,24(3):1-4
[4]陈培基,李刘冬,李来好,等.二种褐藻膳食纤维的功能活化及其生理功能的比较[J].食品科学,2005,26(1):226-230
[5]李刘冬,李来好,陈培基,等.马尾藻袋泡茶的生产工艺与条件[J].食品与发酵工业,2003,29(9):31-34
[6]李来好,吴燕燕.马尾藻袋泡茶的生产技术[J].广州食品工业科技,1995,11(2):50-51
[7]丁恒山.中国药用孢子植物[M].上海:科学技术出版社,1982
[8]曾呈奎,陆保仁.中国海藻志[M].第三卷,第二册.北京:科学出版社,2001
[9]中华人民共和国卫生部药典委员会编.中华人民共和国卫生部药典[M].一部.北京:人民卫生出版社,1990
[10]陈锡林.浙江海洋药用藻类资源调查研究初报[J].浙江中医学院学报,2000,24(2):65-67
[11]崔征,李玉山,肇文荣.中药海藻及数种同属植物的药理作用[J].中国海洋药物,1997,(3):5-8
[12]刘秋英,孟庆勇.半叶马尾藻多糖体内抗肿瘤作用及其机制探讨[J].第一军医大学学报,2004,24(4):434-436
[13]孟庆勇,刘志辉,郑辉.半叶马尾藻多糖对辐射损伤小鼠骨髓细胞集落生成的影响[J].青岛海洋大学学报,1997,35(3):503-506
[14]Na H J,Moon P D,Ko S G,et al.Sargassum hemiphyllum inhibits a topic reaction via the regulation of inflammatory mediators[J].J Pharmacol Sci,2005,97(2):19-26
[15]严小军.马尾藻碘化学形态研究突破(南方产马尾藻碘含量最高)[J].海洋科学,2000,24(3):56
[16]韩丽君,范晓,周天成,等.广西北部湾马尾藻脂肪酸组成的研究[J].海洋科学集刊,1995,36:175-180
[17]蔡双莲,李敏.多不饱和脂肪酸的研究进展[J].生命科学研究,2003,7(4):289-304
[18]Yeh S T,Lee C S,Chen J C.Administration of hot-water extract of brown seaweed Sargassum duphcatun via immersion and injection enhances the immune resistance of white shrimp Litopenaeus vannamei[J].Fish Shellfish Immunol,2006,20(3):32-45
[19]常巧玲,孙建义.海藻饲料资源及其在水产养殖中的应用研究[J].饲料工业,2006,27(2):62-64
[20]王如才,于瑞海.海藻榨取液在海湾扇贝亲贝蓄养中的应用[J].海洋科学,1989,6:55-56
[21]潘鲁青,张涛.海藻磨碎液饲育中国对虾幼体试验[J].海洋科学,1997,2:3-5
[22]Chotigeata W,Tongsupa S,Supamataya K,et al.Effect of Fucoidan on Disease Resistance of Black Tiger Shrimp[J].Aquaculture,2004,233(1-4):23-30
[23]周歧存,赵华超.海藻在罗氏沼虾饲料中的应用研究[J].饲料研究,2001(8):5-8
[24]周歧存,肖风波.海藻在南美白对虾饲料中的应用研究[J].海洋科学,2003,27(3):66-69
[25]Bindu M S,Sobha V.Conversion efficiency and nutrient digestibility of certain seaweed diets by laboratory reared Labeorohita(Hamilton)[J].Indian J Exp Biol.2004,42(12):39-44
[26]赵学武,徐鹤林.关于马尾藻代替部分粮食和矿物质饲喂肉鸡的探讨[J].青岛海洋大学学报,1990,20(1):86-91
[27]张善明,刘强,张善垒.从海带中提取高粘度海藻酸钠[J].食品工业科技,2002,(3):72-73
[28]侯振建,刘婉乔.从马尾藻中提取高粘度海藻酸钠[J].海洋科学,1997,(5):18-20
[29]邹荣岳.从马尾藻中生产海藻酸钠[J].广西科学学院学报,1999,(2):81-83
[30]何培民,徐姗楠,张寒野.海藻在海洋生态修复和海水综合养殖中的应用研究简况[J].渔业现代化,2005(4):15-16
[31]Tsui M T,Cheung K C,Tam N F.A comparative study on metal sorption by brown seaweed[J].Chemosphere.,2006,65(1):1-7
[32]杨宇峰,宋金明,林小涛.大型海藻栽培及其在近海环境的生态作用[J].海洋环境科学,2005,24(2):77-80
[33]Terawali T,Yoshikawa K,Yoshida G.Ecology and restoration techniques for Sargassum beds in the Seto Inland Sea,Japan[J].Mar Pollut Bull,2003,47:198-201
[34]Crouch I J,Beckett R P,Van Stadan.J.Effects of seaweed concentrate on the growth and mineral nutrient stressed lettuce[J].J.Applied Phycology,1990,2:269-272
[35]汤学军,潘列梅,刘慧玲.马尾藻活性物质对水稻幼苗抗旱性的作用[J].海洋科学,2004(10):45-47
[36]Sivasankari S,Venkatesalu V,Anantharaj M.Effect of seaweed extracts on the growth and biochemical constituents of Vigan sinensis[J].Bioresour Technol.2006,97(14):45-51
[37]王平.褐藻胶在日用化学品中的应用[J].现代日用科学,2002,(5):44
[38]张惟杰主编.糖复合物生化研究技术[M].浙江大学出版社,第二版,2003
[39]庞启深,郭宝江,阮继红.螺旋藻多糖对核酸内切酶活性和DNA修复合成的增强作用[J].遗传学报,1988,15:374-381
[40]周慧萍,蒋巡天,陈琼华等.浒苔多糖的降血脂及其对SOD活力和LPO含量的影响[J].生物化学杂志,1995(11):161-165
[41]徐旭,于冰,汤立达,等.海洋生物多糖的药用功能[J].天津药学,2004,16(6):51-53
[42]薛静波,刘希英,张鸿芬.海带多糖对小鼠腹腔巨噬细胞的激活作用[J].中国海洋药物,1999,18(3):23-25
[43]Okai Y,Ishizaka S,Higashi-Okai K,et al.Detection of immunomodulating activities in an extract of Japanese edible seaweed,Laminaria japonica[J].J Sci Food Agric,1996,72(4):455-460
[44]Zvyagintseva,Tatiana N.Inhibition of complement activation by water-soluble polysaccharides of some fareastern brown seaweeds[J].Comp Biochem Physiol,Part C:Toxicol Pharmacol,2000,126C(3):209-215
[45]苗本春,耿美玉,李静,等.海洋硫酸多糖911免疫增强作用的探讨[J].中国海洋药物,2002,21(5):1-4
[46]黄益丽,郑忠辉,苏文金等.二色桌片参的化学成分研究:Ⅲ.二色桌片参多糖-1-岩藻聚糖的免疫调节[J].海洋通报,2001,20(1):88-91
[47]陈文星,吴皓,金亦涛.珠蚌多糖的免疫调节作用研究[J].中药药理与临床,2001,17(5):16-18
[48]朱爱民,扬晓峰,李晟刚,等.螺旋多糖对小鼠免疫系统的作用[J].山东医药工业,2001,20(6):44-45
[49]陈绍瑗,莫卫民,潘远江.海洋药物研究(Ⅲ)——羊栖菜多糖[J].兰州大学学报(自然科学版),1998,34(4):110-113
[50]徐明芳,高孔荣,刘婉乔.海藻保健功能的研究进展[J].食品科学,1995,16(12):19-22
[51]汪开治.海藻含有免疫抑制物[J].生物学通报,1995,30(1):11
[52]姚福汉,蒋伯诚.海藻中具有免疫赋活作用的多糖[J].中国野生植物资源,1997,16(3):6-7
[53]王庭欣,秦淑贞,赵文,等.海藻多糖对环磷酰胺诱发小鼠骨髓微核率的抑制作用[J].癌变·畸变·突变,1999,11(2):106-107
[54]王庭欣,蒋东升,赵文,等.海带多糖对小鼠免疫功能的影响[J].中国老年学杂志,1999,19(专刊):12
[55]徐惠,于志洁,等.螺旋藻黏多糖的分离及其免疫学作用[J],中国生化药物杂志,1997,18(2):72-74
[56]Li J Z,Bao C X,Chen G Z.Antithrombin activity and platelet aggregation by acid mucopolysaccharides isolated from stichopus japonicus selenka.China[J].Acta Pharmacologica Sinica,1985,6(2):107-110
[57]马西,Lindhout T,Beguin S,et al.刺参酸性粘多糖抑制凝血酶活性的辅因子[J].中华血液学杂志,1990,11(5):241-243
[58]Nagase H,Enjyoji K I,Minamiguchi K,et al.Depolymerized holothurian glycosaminoglycan with novel anticoagulant actions:antithrombin Ⅲ-and heparin cofactor Ⅱ-independent inhibition of factor X activation by factor LXa-factor Ⅷa complex and heparin cofactorⅡ-dependent inhibition of thrombin[J].Blood,1995,85:1527-1534
[59]王学锋,李志广,储海燕,等.海参糖胺聚糖抗血栓形成机制的研究[J].中国新药与临床杂志,2002,21(12):718-721
[60]刘志峰,宫晓黎,魏淑贞.五种海藻多糖体外抗血小板聚集作用观察[J].中国海洋药物,2001,20(2):36-38
[61]辛现良,耿美王,戚欣,等.新型抗脑缺血海洋新药989作用机理的初步探讨[J].青岛海洋大学学报,2001,31(4):536-540
[62]耿美玉,辛现良,李静,等.新型海洋硫酸多糖DPS对大鼠脑缺血的保护作用[J].海洋与湖沼,2001,32(2):192-197
[63]刘秋英,孟庆勇,刘志辉.海藻多糖抗肿瘤作用的研究进展[J].中国海洋药物,2003,22(4):45-48
[64]曲显俊,崔淑香,解砚英.螺旋藻多糖抗癌作用的实验研究[J].中国海洋药物,2000,19(4):10-14
[65]许东晖,许实波,王兵等.皱纹盘鲍多糖抗肿瘤药理作用研究[J].热带海洋,1999,18(4):86-90
[66]Ellouali M,Boisson V C,Durand P,et al.Antitumor activity of low molecular weight fucans extracted from brown seaweed Ascophyllum nodosum[J].Anticancer Res,1993,13:2011-2020
[67]Mishima T,Murata J,Toyoshima M,et al.Inhibition of tumor invasion and metastasis by calcium spirulan(Ca-SP),a novel sulfated polysaccharide derived from a blue-green alga,Spirulina platensis[J].Clinical and Experimental Metastasis,1998,16(6):541-550
[68]卢睿春,侯振建,刘婉娇.亨氏马尾藻硫酸多糖抗肿瘤活性的研究[J].海洋科学,1998,3:63-64
[69]曾波航,王光明,曾亚仑.钝顶螺旋藻多糖对急性白血病病人NK细胞的作用[J].中国海洋药物,2000,(6):45-47
[70]寿佩勤.部分海藻多糖的抗肿瘤作用研究和临床应用[J].现代实用医学,2004,16(7):432-434
[71]陈毓强,吴炳南.四种多糖化合物的抗癌作用及其对免疫功能的影响[J].癌症,1997,16(3):198-200
[72]张朝晖,蔡宝昌.海洋药物研究与开发[M].北京:人民卫生出版社,2003
[73]耿美玉,辛现良,管华诗.海洋硫酸多糖类药物911抗艾滋病毒作用及其机理研究[J].中国药理学会通讯,2001,18(4):13-23
[74]于红,张学成.螺旋藻多糖抗HSV-1作用的体外实验研究[J].高技术通讯,2002,12(9):65-69
[75]Albertus J S.Medicinal and pharmaceutical uses of seaweed natural products:A review[J].Journal of Applied Phycology,2004,16:245-262
[76]魏文青,王明霞,丛建波,等.海藻硫酸多糖抗乙型肝炎病毒的实验研究[J].中华肝脏病杂志,2002,10(2):112
[77]王秉及.海洋药物的抗病毒研究[J].生物工程进展,1996,16(6):34-36
[78]王安利,胡俊荣.海藻多糖生物活性研究进展[J].海洋科学,2002,26(9):36-39
[79]徐明芳,高孔荣,刘婉乔.海藻保健功能的研究进展[J].食品科学,1995,16(12):19-22
[80]王炳岩,季宇彬.海藻多糖对白血病L615小鼠LPO含量及GR、GSH—PX、CAT、SOD酶活性的影响[J].中医药信息,1994,11(5):43-45
[81]李妍,田晓华,丛建波,等.海藻多糖抑制白细胞呼吸爆发作用研究[J].生物化学与生物物理进展,1999,26(2):162-164
[82]刘凤艳,钟红茂,范洁伟,等.海藻多糖药理作用研究新进展[J].广东药学,2005,15(3):81-84
[83]Kumar G P,Suheesh S,Vijayalakshmi N R.Hypoglyeemic effect of coecinia indica,mechanism of action[J].Planta Med,1993,59(4):330-332
[84]侯建明,蓝进.海洋生物多糖(AC-1)防治高脂血症的实验报告[J].海军医学杂志,2001,22(3):197-199
[85]王远红,徐家敏,耿美玉,等.海洋硫酸多糖916对实验性高脂血症大鼠血浆中含硫氨基酸的影响[J].中国海洋药物,2000,19(4):23-24
[86]王兵,李靖,马舒冰,等.羊栖菜多糖降血糖的实验研究[J],中国海洋药物,2000,3:33-35
[87]李八方,毛文君,曹立民.羊栖菜多糖对高血脂模型大鼠血脂的影响[J].中国水产科学,2000,7(2):56-58
[88]周慧萍,陈琼华.紫菜多糖的抗凝血和降血脂作用[J].中国药科大学学报,1990,21(6):358-360
[89]邓槐春,谈竞光,谢娇娥等.海藻多糖对高血脂症的临床疗效初步观察[J].中草药,1987,18(8):12
[90]阮积惠.海藻主要药用成分的研究和展望[J].东海海洋,2001,19(2):1-9
[91]范晓,周永航.海藻中的甜菜碱及其类似物[J].海洋科学,1999,23(3):24-26
[92]张成武,曾昭琪,张嫒贞,等.钝顶螺旋藻多糖和藻蓝蛋白对小鼠急性放射病的防护作用[J].营养学报,1996,3:327-331
[93]李德远,王海滨,张声华,等.海带岩藻糖胶及褐藻胶抗辐射效应研究[J].武汉食品工业学院学报,1999,2:18-22
[94]王友顺,黎露刚,吴侃,等.螺旋藻多糖对给予环磷酰胺BALB/C小鼠的保护作用[J].海洋科学,1997,6:36-38
[95]王友顺,黎露刚,吴侃,等.螺旋藻多糖对CD3AK细胞增殖能力的影响[J].海洋科学,1998,3:37-39
[96]王庭欣,秦淑贞,赵文,等.海带多糖对环磷酰胺诱发小鼠骨髓细胞微核率的抑制作用[J].癌变.畸变.突变,1999,11(2):106-107
[97]田晓华,丛建波,施定基,等.褐藻多糖硫酸酯清除活性氧自由基作用及动力学的ESR研究[J].营养学报,1997,19(1):32-37
[98]藤霞,丛建波,田晓华,等.海藻硫酸多糖抗氧化与抗肿瘤作用的实验研究[J].营养研究,1998,20(1):48
[99]詹林盛,张新生,吴小红,等.褐藻多糖对氧化应激诱导的淋巴细胞DNA损伤的保护作用[J].中国海洋药物,1999,4:4-7
[100]石达友,刘汉儒,黎建华.中药对鸡新城疫灭活疫苗免疫效果的影响[J].中国兽医科技,2004,34(2):42-46
[101]李子昆.海藻提物的减肥及保湿作用研究[J].四川日化,1997,(1):12-15
[102]诸葛健,赵振锋,方慧英.功能性多糖的构效关系[J].无锡轻工大学学报,2002,21:209-212
[103]Ishibashi K,Miura N N,Adachi Y,et al.Relationship between solubility of grifolan,a fungall,3-β-D-glucan,and production of tumor necrosis factor by macrophages in vitro[J].Biosci Biotechnol Biochem,2001,65(9):1993-2000
[104]田庚元,冯宁澄.多糖类免疫调节剂的研究和应用[J].化学进展,1994,6(2):114-124
[105]王长云,管华诗.多糖抗病毒研究进展Ⅱ:硫酸多糖抗病毒作用[J].生物工程进展,2000, 20:3-8
[106]Ehresmann D W,Deig E F,Hatch M T,et al.Dextran sulfate,a potent anti-HIV agent in vitro having synergism with sidovudine[J].Lancet,1987,1:1379-1386
[107]李艳辉,王琦.真菌多糖的生物活性与构效关系研究评价[J].吉林农业大学学报,2002,24(2):70-77
[108]王兆梅,李琳,郭祀远,等.活性多糖构效关系研究评述[J].现代化工,2002,22(8):18-22
[109]王超英,吕金顺,邓芹英.菌类多糖的生物活性与结构关系[J].天水师范学院学报,2001,21(5):18-21
[110]杜巍,李元瑞,袁静.食药用茵多糖生物活性与结构的关系[J].食用菌,2001,(2):3-5
[111]方唯硕,刘相.具有抗HIV活性的天然产物[J].国外医学:中医中药分册,1993,8(2):61-69
[112]Saito H,Yoshioka Y,Uehara N,et al.Relationship between conformation and biological response for(1→3)-β-D-glucans in the activation of coagulation Factor G from limulus amebocyte lysate and host-mediated antitumor activity.Demonstration of single-helix conformation as a stimulant [J].Carbohydrate Research.,1991,217:181-190
[113]周庆华.超临界萃取技术最佳方法的研究[J].哈尔滨理工大学学报,2003,8(3):125-126
[114]何江川,韩永萍.超滤膜分离法在多糖分离提取中的应用[J].食用菌,2005,1:5-7
[115]赵晓燕,王长云.海洋多糖分子修饰方法研究概况[J].海洋科学,2000,24:20-22
[116]Sakai T,Kimura H,Kojima K,et al.Marine bacterial sulfated fucoglucuronomannan(SFGM)lyase digests brown algal SFGM into trisaccharides[J].Mar Biotechnol(NY),2003,5:70-78
[117]Miller I J,Blunt J W.Desulfation of algal galactan[J].Carbohydrate Research,1998,309:39-43
[118]梁忠岩,苗春艳,张翼伸,等.化学修饰对斜顶菌多糖抑瘤活性影响的研究[J].中国药学杂志,1996,30:613-615
[119]Uryu T,Ikushima N,Katsuraya K,et al.Sulfated alkyl oligoaacharides with potent inhibitory effects on HIV infection[J].Biochem Pharmacol,1992,43:2385-2392
[1]卢群,丘泰球,胡爱军.超声强化法提取海藻油的研究[J].广东药学院学报,2003,19(2):104-105
[2]张秀妍,姜爱莉,王长海.玻璃海鞘脂肪酸提取工艺的研究[J].海洋通报,2004,23(4):93-96
[3]Jamieson G R,Reid E H.The component acids of some marine algal lipids[J].Phytochemistry,1972,11:1423-1432
[4]刘志伟.超临界流体萃取技术及其在食品工业中的应用[J].食品研究与开发,2004,25(2):3-6
[5]刘雄,阚建全.超临界二氧化碳萃取和精制柚皮香精油的研究[J].食品与机械,2003,(4):13-14
[6]周庆华.超临界萃取技术最佳方法的研究[J].哈尔滨理工大学学报,2003,8(3):125-126
[7]颜英.超临界CO_2萃取沙棘油的研究[J].精细石油化工,2003,(3):39-41
[8]Annadurai G.Design of optimum reSPPonse surface experiments for adsorption of direct dye on Ehitosan[J].Bioproc Eng,2000,23:451-455
[9]Reddy P R M,Reddy G,Seenayya G.Produetion of the nnostable pullulanase by Clostridium thermosulfurogenes SV2 in solid-state fermentation:optimization of nutrients levels using reSPPonse surface methodology[J].Bioproc Eng,1999,21:497-503
[10]Rastogi N K,Rashmi K R.Optimisation of enzymatic liquefaction of mango pulp by reSPPonse surface methodology[J].Eur Food Res Technol,1999,209:57-62
[11]Gouveia E R,Baptista-Neto A.Optimisation of medium composition for clavulanic acid production by streptomyces clavuligerus[J].Biotechnol Lett,2001,23:157-161
[12]Ambati P,Ayyanna C.Optimizing medium constituents and fermentation conditions for citric acid production from Palmyra jaggery using reSPPonse surface method[J].World J Microbiol Biolotechnol,2001,17:331-335
[13]Chen Q H,He G Q,Mokhtar A M A.Optimization of medium composition for the production of elastase by Bacillus SPP.EL31410 with reSPPonse surface methodology[J].Enzyme Microbial Technol,2002,30:667-672
[14]Bradford M M.A rapid and sensitive method for the quantitation of microgram quantitatives of protein utilizing the principle of protein-dye binding[J].Anal Biochem,1976,72:248-254
[15]冀颐之,杜连祥.深层培养裂褶菌胞外多糖的提取及结构研究[J].微生物学通报,2003,30(5):15-20
[16]高居易,卓建亭,黄一红.紫孢侧耳多糖的提取、纯化及性质的研究[J].福建师范大学学报(自然科学版),1999,15(2):83-87
[17]何江川,韩永萍.超滤膜分离法在多糖分离提取中的应用[J].食用菌,2005,1:5-7
[18]张宁,彭志英.中空纤维超滤膜浓缩胞外多糖PS-9415发酵液的研究[J].Science and Technology of Food Industry,2003,24(2):24-27
[19]刘茉娥等编.膜分离技术[M].化工出版社:1998年第1版
[20]Dubois M,Crilles KA,Hamilton J K,et al.Colorimetric method for determination of sugars and related substances.Analytical Chemistry,1956,28:350-356
[21]陈均辉,陶力,李俊,等.生物化学实验[M].北京:科学出版社,2003
[1]Yamaguchi F,Ota Y,Hatanaka C.Extraction and purification of pectic polysaccharides from soybean okara and enzymatic analysis of their structures[J].Carbohydrate Polymers,1996,30:265-273
[2]Yu R M,Wang L,Zhang H,et al.Isolation,purification and identification of polysaccharides from cultured Cordyceps militaris.Fitoterapia,2004,75:662-666
[3]赵永芳.生物化学技术原理及应用[M].北京:科学出版社,2002
[4]方积年.多糖的分离纯化及其纯度鉴别与分子量测定[J].药学通报,2003,19(10):46-49
[5]李波,许时婴.羊栖菜褐藻糖胶的分级纯化和结构分析[J].无锡轻工大学学报,2004,23(2):86-89
[6]刘敦华,谷文英.沙蒿籽胶多糖的分级纯化和结构分析[J].食品科学,2007,28(3):73-76
[7]吴亚林,黄静,潘远江.无花果多糖的分离、纯化和鉴定[J].浙江大学学报:理学版,2004,31(2):177-179
[8]闻吉昌,催春月,张奕,等.芦荟多糖的分离纯化及结构分析[J].高等学校化学学报,2003,24(7):1189-1192
[9]张金亭,黄勇,李金话,等.小刺猴头多糖I的分离纯化及组成分析[J].中国医学杂志,2005,40(7):545-547
[10]Lloyd A G,Dodgson K S,Price RG,et al.Polysaccharide sulphates[J].Biochimica et Biophysica Acta,1961,1:108-115
[11]Bitter T,Muir H M.Amodified uornic acid carbazole reaction[J].Analytical Biochemistry 1962,(4):330-334
[1]Yoshiaki F,Youichi S,Ko-ichi O.Selective tumoricidal effect of soluble proteoglucan extracted from the basidiomycete,Agaricus blazei Murill,mediated via natural killer cell activation and apoptosis [J].Cancer Immunol Immunother,1998,46:147-159
[2]Yoshiaki F.Tumor-SPPecific cytocidal and immunopotentiating effects of relatively low molecular weight products derived from the Basidiomycete,Agaricus blazei Murill[J].Anticancer Res,1999,19:113-118
[3]毛俊好.白术多糖对小鼠淋巴细胞功能的调节[J].免疫学杂志,1996,12(4):233-236
[4]向道斌,李晓玉.牛膝多糖的抗肿瘤活性及其免疫增强作用(英文)[J].中国药理学报,1993,14(6):556-561
[5]Adachi Y,Okazaki M,Ohno N,et al.Enhancement of cytokine porduction by macrophages stimulated with 口(1口 3)-D-giucan,grifolan(CRN),isolated from Grifola frondosa[J].Biological and Pharmacentical Bulletin,1994,17(12):1554-1560
[6]韩锐主编.抗癌药物研究与实验技术[M].北京:北京医科大学协和医科大学联合版社,1997
[7]Ito H,Shimura K,Itoh H,et al.Antiutmor effects of a new polysaccharide-protein complex(ATOM)prepared from Agaricus blazei(Iwade srtain101)"Himematsutake" and its mechanisms in tumor-bearing mice[J].Anticancer Res,1997,17:277-284
[8]王葡文主编.实验肿瘤学基础[M].北京:人民卫生出版社,1992:279-300
[9]张尔贤,俞丽君.鼠尾藻多糖清除氧自由基作用的研究[J].中国海洋药物,1997,3:1-4
[10]于广利,吕志华,王曙光,等.海黍子提取物对不饱和脂质抗氧化作用[J].青岛海洋大学学报,2000,30(1):75-80
[11]于广利,薛长湖,楼伟风.海藻多糖类化合物对卵磷脂氧化的抑制作用[J].中国水产科学,1998,5(4):48-51
[12]师然新,徐祖洪.青岛沿海九种海藻的类脂及酚类抗菌活性的研究[J].中国海洋药物,1997,4:16-19
[13]Matsukawa R,Dubinsky Z,Kishimoto E,et al.A comparison of screening method for antioxidant activity in seaweeds[J].J Appl Phycol,1997,9:29-35
[14]Nakamura T,Nagayama K,Uchida K,et al.Antioxidant activity of phlorotannins isolated from the brown alga Eisenla bycyclis[J].Fisheries Science,1992,62(6):923-926
[15]石碧,狄莹著.植物多酚tM].北京:科学出版社,2000,124-134
[16]Yan X J,Li X C,Zhou C X,et al.Prevention of fish oil rancidity by phlorotannins from Sargassum kjellmanianum[J].J.Appl.Phycol.,1996,8:201-203
[17]Leong L P,Shui G.An investigation of antioxidant capacity of fruits in Singapore markets[J].Food Chemistry,2002,76(1):69-75
[18]Benzie I F,Strain J J.The ferric reducing ability of plasma(FRAP) as a measure of"antioxidant power":the FRAP assay[J].Journal of Analytical Biochemistry,1996,239(1):70-76
[19]Yen G C,Chen H Y.Antioxidant activity of various tea extracts in relation to their antimutagenicity.J Agric Food Chem,1995,43:27-32
[20]Wang J C,Xing G S,Hu W D,et al.Effects of Ge-132 on oxygen free radicals and the lipid peroxidation induced by hydroxyl free radical in vitro.Chinese Pharmaceutical Journal,1994,29:23-25
[21]Nurmi K,Ossipov V,Haukioja E,et al.Variation of total phenolic content and individual low-molecular-weight phenolics in foliage of mountain birch trees(Betula pubescens sSPP.tortuosa)[J].J Chem Ecol,1996,22:2023-2040
[22]Decker E A,Welch B.Role of ferritin as a lipid oxidation catalyst in muscle food.J Agric Food Chem,1990,38:674-677
[23]Dias P F,Siqueira J M,Vendrascolo L F,et al.Antiangiogenic and antitumoral properties of a polysaccharide isolated from the seaweed Sargassum stenophyllum[J].Cancer Chemotherapy and Pharmacology,2005,56(4):436-446
[24]Duarte M E R,Cardoso M A,Noseda M D,et al.Structural studies on fucoidans from the brown seaweed Sargasswn stenophyllum[J].Carbohydrate Research,2001,333(4):281-293
[25]王长云,管华诗.糖抗病毒作用研究进展Ⅱ硫酸多糖抗病毒作用[J].生物工程进展,2000,20(2):3-8
[26]Zhang M,Zhang L,Cheung P C K,et al.Molecular weight and anti-tumor activity of the water-soluble polysaccharides isolated by hot water and ultrasonic treatment from the sclerotia and mycelia of Pleurotus tuber-regium[J].Carbohydrate Polymers,2004,56:123-128
[27]Hu F L,Lu R L,Huang B,et al.Free radical scavenging activity of extracts prepared from fresh leaves of selected Chinese medicinal plants[J].Fitoterapia,2004,75(1):14-23
[28]Aruoma O I.Free radicals,oxidative stress,and antioxidants in human health and disease.J Am Oil Chem Soc,1998,75:199-212
[29]Shon M Y,Kim T H.Sung N J.Antioxidants and free radical scavenging activity of Phellinus baumii(Phellinus of Hymenochaetaceae) extracts.Food Chem,2003,82:593-597
[30]Jimenez-Escrig A,Jimenez-Jimenez I,Pulido R,et al.Antioxidant activity of fresh and processed edible seaweeds[J].Journal of the Science of Food and Agriculture,2001,81(5):530-534
[31]Yan X,Nagata T,Fan X.Antioxidative activities in some common seaweeds[J].Plant Foods for Human Nutrition,1998,520):253-262
[32]Xue C H,Fang Y,Lin H,et al.Chemical characters and antioxidative properties of sulfated polysaccharides from Laminaria japonica[J].Journal of Applied Phycology,2001,13(1):67-70
[33]Xue C,Yu G,Hirata T,et al.Antioxidative activities of several marine polysaccharides evaluated in a phoSPPhatidylcholine-liposomal suSPPension and organic solvents[J].Bioscicnce,Biotechnology,and Biochemistry,1998,62(2):206-209
[34]Ruperez P,Ahrazem O,Leal J A.Potential antioxidant capacity of sulfated polysaccharidcs from the edible marine brown seaweed Fucus vesiculosus[J].Journal of Agricultural and Food Chemistry,2002,50(4):840-845
[35]Yamaguchi F,Ariga T Yoshimira Y,et al.Antioxidant and anti-glycation of carcinol from Garcinia indica fruit rind.J Agric Food Chem,2000,48:180-185
[1]Miyazaki T,Nishijima M.Studies on fungal polysaccharides.ⅩⅩⅦ.Structural examination of a water-soluble,antitumor polysaccharide of Ganoderma lucidum[J].Chem Pharm Bull,1981,29:3611-3616
[2]Yamada H,Kawaguchi N,Ohmori T,et al.Structure and antitumor activity of an alkali-soluble polysaccharide from Cordyceps ophioglossoides[J].Carbohydr Res,1984,125:107-115
[3]Iino K,Ohno N,Suzuki I,et al.Structural characterization of a neutral antitumorβ-D-glucan extracted with hot sodium hydroxide from cultured fruit bodies of Grifola frondosa[J].Carbohydr Res,1985,141:111-119
[4]Misaki A,Nasu M,Sone Y,et al.Comparison of structure and antitumor activity of polysaccharides isolated from Fukurotake,the fruiting body of Volvariella volvacea[J].Agric Biol Chem,1986,50:2171-2184
[5]王森,丁霄霖.葡聚糖生物活性与结构的关系[J].无锡轻工大学学报,1997,(2):90-94
[6]Ning J,Zhang W H,Yi Y T,et al.Synthesis of β-(1-6)-ranched β-(1-3) glucohexanse and its analogues containing an α-(1-3) linked bond with antitumor activity[J].Bioorganic Med Chem,2003,11:2193-2203
[7]魏远安,方积年.高效凝胶渗透色谱法测定多糖纯度及分子量[J].药学学报,1989,24(7):532-536
[8]Hagel L.Comparison of some soft gels for the molecular-weight distribution analysis of dextran at enhanced flow-rates[J].J Chromatogr,1978,160:59-71
[9]董群,方积年.寡糖及多糖甲基化方法的发展及现状[J].天然产物研究与开发,1995,7(2):60-65
[10]徐桂芸,常理文.微量寡糖的甲基化分析-用于魔芋葡甘多糖酶解产物的结构测定[J].分析化学,1996,24(12):1400-1404
[11]Hakomori S.A rapid permethylation of glycolipid,and polysaccharide catalyzed by methylsulfinyl carbanino in dimethyl sulfoxide[J].J Biochem,1964,55:205-208
[12]Miyazaki T,Oikawa N,Yamada H,et al.Structural examination of antitumour,watersoluble glucans from Grifora umbellate by use of four types of glucanase[J].Carbohydr Res,1978,65:235-243