荔枝果肉多糖的分离纯化与结构表征及抗氧化活性研究
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摘要
多糖广泛存在于植物、动物和微生物体内,研究证明许多多糖具有提高免疫力、降血糖、降血脂、抗氧化、抗肿瘤、抗病毒等多种生物活性。荔枝(Litchi chinensis Sonn)原产于我国,属无患子科,俗有“岭南果王”之称,为广东四大名果之一。目前关于荔枝果肉多糖的研究不多,不够深入。为开展荔枝精深加工,本课题对不同品种荔枝果肉多糖含量差异,荔枝干加工方法对果肉多糖活性的影响,荔枝多糖的提取、分离纯化及抗氧化护肝功能进行了系统的研究。
本课题选取广东省主栽的21个荔枝品种,测定其水溶性多糖含量,比较得出多糖含量最高的品种为黑叶,含量达到15.72g/kg,由此选定黑叶为提取多糖及多糖结构和生物活性研究材料。
比较5种加工荔枝干方法(晒干、热风干燥、微波干燥、真空干燥和冷冻干燥),对荔枝多糖含量及抗氧化活性的影响,研究发现各种干燥方法对荔枝果肉多糖含量和抗氧化活性影响都较小,热风干燥法烘干产量大,设备简单,产品质量较好,所以选择热风干燥法制备实验用荔枝干。
在荔枝多糖提取工艺实验中,分别研究荔枝多糖的热水浸提、微波辅助提取和超声辅助提取的优化工艺条件,结果表明三种提取方法的多糖提取率分别为热水浸提24.22%、微波提取24.26%、超声提取24.32%,统计分析表明差异不显著;提取时间长短依次为:热水浸提>超声提取>微波提取。自由基清除能力测定结果表明,各种提取方法对荔枝果肉多糖抗氧化活性影响较小。荔枝多糖三种提取方法比较表明微波法提取率较高,时间较短,多糖活性不受提取方式影响,所以在后续研究中采用微波辅助提取法提取多糖。
建立不同乙醇浓度沉降制备荔枝多糖组分的方法。将多糖溶液浓缩到浓度为2%左右,用95%乙醇调节体系乙醇浓度为20%,4℃下醇沉12小时,得到荔枝粗多糖沉淀LFP1;上清液真空浓缩到多糖浓度为2.0%左右,加入95%乙醇调节体系的乙醇浓度到50%,4℃下醇沉12小时,得粗多糖沉淀LFP2;按相同方法,加入95%乙醇调节体系的乙醇浓度到80%,得粗多糖沉淀LFP3。另外采用一次性沉降法,用80%乙醇沉降可得多糖LFP4。四种荔枝果肉多糖LFP1、LFP2、LFP3、LFP4的沉降得率分别为6.4%、53.3%、37.4%和95%。
采用DEAE-52对荔枝果肉多糖组分LFP_2进行分离,得到三个级分:LFPC_1、LFPC_2和LFPC_3;选取多糖量较高的级分LFPC_2和LFPC_3,用Sephadex G-100凝胶柱进一步分离纯化,LFPC_2过柱后分出两个级分:LFPC_2a和LFPC_2b,后者多糖量较少;LFPC_3过G-100凝胶柱后,只有一个级分。选取两个多糖级分:LFPC_2a和LFPC_3,用纸层析和凝胶层析法对LFPC_2a和LFPC_3进行纯度鉴定,证明这两个级分比较均一。
气相色谱等方法测定多糖组成,结果显示:LFPC_2a级分主要由阿拉伯糖、鼠李糖和葡萄糖组成,摩尔比为:1.00:1.37:1.21;经红外光谱,结合高碘酸氧化及Smith降解分析,显示多糖LFPC_2a存在1→2键、1→3键和1→6键,不存在1→4键,且1→3键、1→2键、1→6键之比约为:63.84:30.16:6.0。渗透凝胶色谱测定LFPC_2a的峰位分子量为43927 Da。多糖LFPC_2a粒度分布测定结果显示,不同pH条件下多糖聚集体大小顺序分别为:酸性>中性>碱性,粒度分布测定结果还表明,无论是何种条件,荔枝多糖在溶液中分布都是不均一的。LFPC_2a的X-Rays衍射分析结果显示荔枝多糖中含结晶性的多糖极少,结晶性很差,为无定形物质。扫描电镜研究发现,荔枝果肉多糖LFPC_2a在放大倍数较高的情况下,图片显示多糖表面紧密,较平整,这说明荔枝多糖分子间相互作用较强,结合紧密。热力学分析表明,与在氮气中相比,多糖LFPC_2a在空气中的热重变化规律与其相似,但变化更剧烈、更不稳定。
LFPC_3级分主要由阿拉伯糖、鼠李糖,少量半乳糖和葡萄糖组成,摩尔比为:2.42:3.44:1.00:1.65。根据洗脱曲线、茚三酮反应和紫外扫描结果,LFPC_3级分中存在少量蛋白质,LFPC_3可能是一种糖-蛋白结合物,经自动氨基酸分析仪分析显示LFPC_3由17种氨基酸组成,氨基酸总量为2626.87mg/100g。渗透凝胶色谱测定LFPC_3峰位分子量为33723 Da。
通过体外抗氧化实验,研究4种荔枝多糖LFP1、LFP2、LFP3、LFP4的抗氧化活性,结果表明:4种多糖组分均能清除DPPH自由基、超氧阴离子自由基、羟基自由基,且呈现一定的量效关系;各级分表现出很好的铁离子螯合能力,各组分的还原力随着多糖浓度的增加而提高。在各级分中LFP2抗氧化活性较好,所以选LFP2进行动物实验。通过CCl_4和酒精肝损伤小鼠模型,探讨荔枝多糖LFP2的抗氧化护肝作用,结果表明在CCl_4诱导急性肝损伤实验中,LFP2可以提高肝脏和血清中的SOD活性,并降低MDA含量,且呈一定的量效关系;同时LFP2可以提高小鼠肝脏中CAT和GSH-Px活性,从而提高肝脏的抗氧化能力,CCl_4模型组的小鼠血清中ALT、AST活性升高非常明显,LFP2中、高剂量给药组酶抑制率与阳性对照组相接近。在酒精诱导急性肝损伤实验中,荔枝多糖LFP2三个剂量组ALT、AST活性与模型组相比差异都达到了极显著水平(P<0.01),荔枝多糖中、高剂量给药组酶抑制率与阳性对照组相接近,说明荔枝多糖对酒精肝损伤有一定的保护作用。
Polysaccharide is widely found in plants, animals and microorganisms. Polysaccharides have functions of modulating immunity, reducing blood sugar and blood fat, antioxidation, anti-tumor, antivirus et al. Litchi (Litchi chinensis Sonn ) originated from China, called‘king of fruit’in South China, is one of four famous fruits in Guangdong province. Little research has been reported on polysaccharide of litchi pulp in relation to its biological activity. To further develop litchi fruits processing, studies were carried on isolation and purification of polysacchride from pulp tissue of litchi, and its antioxidant activity on liver protection was also estimated.
Twenty-one litchi cultivars were choosen in this project. The water-soluble polysaccharide content was determined. The results showed that Heiye had the higest polysaccharide content with the value of 15.72g/kg. Thus, Heiye was selected for the following trails.
Five different desication methods were used to produce litchi nuts. The results showed that the polysaccharide content and antioxidant activity of litchi polysaccharide were steady. In this project, we choose oven-drying to produce litchi nuts.
The extraction methods of litchi were systematacially studied,the methods of leaching with hot water, microwave-assisted extraction and ultrasonic-assisted extraction were investigated, respectly. The results showed that: the extraction yield of litchi extracted by hot water was 24.22%, the extraction yield of extracted by microwave-assisted was 24.26%, the extraction yield of extracted by ultrasonic-assisted was 24.32%. The extraction time of three extraction methods was different: hot water> ultrasonic-assisted> microwave-assisted. Three extraction methods had no effect on antioxidant activity of litchi polysaccharide. In this project, microwave-assisted extraction was choosen to extract the litchi polysaccharide.
Precipitation of litchi polysaccharides with different ethanol concentration: LFP1 was obtained by precipitation extracts with 20% ethanol solution. The supernatant was added ethanol to concentration of 50% to precipitate LFP2. Then the supernatant was added ethanol to concentration of 80% to precipitate LFP3. LFP4 was obtained by precipitation extracts with 80% of ethanol solution directly. Then, LFP1, LFP2, LFP3 and LFP4 were gained after purification.
LFP2 was chromatographed on a DEAE-52 anion-exchanging column to yield two major fractions:LFPC2 and LFPC3. LFPC2 and LFPC3 were subjected to further purification on a Sephadex G-100 gel filtration column. Two major fractions, LFPC_2a and LFPC_3 were collected.
By analysis of gel permeation chromatography (GPC) and gas chromatography (GC), LFPC_2a, which had a average molecular weight of 43927 Da, composed of arabinose, ribose, galactose and glucose in the molar ratio of 1.67:2.15:1.54:1.00.The results of periodate oxidation and Smith degradation showed that the molar percentage ratio of (1→3)-glycosidic linkages, (1→2)-glycosidic linkages and (1→6)-glycosidic linkages was about 63.84%:30.16%:6.0%. By using the LSCM to study LFPC_2a behavior in the solution, the conformation could be changed by the pH value. By SEM, LFPC_2a was observed the smooth figure under high-powered microscope. Thermal analysis of LFPC_2a indicated that litchi polysaccharide had similar endothermic peaks in nitrogen and air.
LFPC_3, which had a average molecular weight of 33723 Da, composed of arabinose, rhamnose, galactose and glucose in the molar ratio of 2.42:3.44:1.00:1.65. The polysaccharide absorption peak showed similar time and shape as the protein absorption peak, suggesting that LFP3 is likely a glycoprotein.
In vitro antioxidant activity of LFP1, LFP_2, LFP3 and LFP4 showed that exhibited a dose-dependent free radical scavenging activity against DPPH radical, superoxide anions, hydroxyl radical, chelating ability and reducing power. Among the four different fractions, LFP_2 showed the strongest scavenging activity.
The antioxidant activity of litchi polysaccharide LFP_2 for mouse liver protection was investigated by CC14-induced and ethanol-induced liver injury experiments. Results indicated that LFP_2 increased SOD activity of mouse liver and serum, and reduced the content of MDA by dose-dependent pattern. LFP_2 also increased mouse liver CAT and GSH-Px activities. In the experiment of CC14-induced mouse oxidative injury, ALT and AST activities of mouse liver increased significantly, and high and medium dossage LFP_2 could inhibit the enzyme activities of ALT and AST. Results of experiment of ethanol-induced mouse oxidative injury showed that, three dossage of LFP_2 could also inhibit the enzyme activities of ALT and AST. These results indicated that litchi polysaccharide could increase the activity of antioxidant enzymatic system and antioxidant ability of liver to protect from injury.
本课题选取广东省主栽的21个荔枝品种,测定其水溶性多糖含量,比较得出多糖含量最高的品种为黑叶,含量达到15.72g/kg,由此选定黑叶为提取多糖及多糖结构和生物活性研究材料。
比较5种加工荔枝干方法(晒干、热风干燥、微波干燥、真空干燥和冷冻干燥),对荔枝多糖含量及抗氧化活性的影响,研究发现各种干燥方法对荔枝果肉多糖含量和抗氧化活性影响都较小,热风干燥法烘干产量大,设备简单,产品质量较好,所以选择热风干燥法制备实验用荔枝干。
在荔枝多糖提取工艺实验中,分别研究荔枝多糖的热水浸提、微波辅助提取和超声辅助提取的优化工艺条件,结果表明三种提取方法的多糖提取率分别为热水浸提24.22%、微波提取24.26%、超声提取24.32%,统计分析表明差异不显著;提取时间长短依次为:热水浸提>超声提取>微波提取。自由基清除能力测定结果表明,各种提取方法对荔枝果肉多糖抗氧化活性影响较小。荔枝多糖三种提取方法比较表明微波法提取率较高,时间较短,多糖活性不受提取方式影响,所以在后续研究中采用微波辅助提取法提取多糖。
建立不同乙醇浓度沉降制备荔枝多糖组分的方法。将多糖溶液浓缩到浓度为2%左右,用95%乙醇调节体系乙醇浓度为20%,4℃下醇沉12小时,得到荔枝粗多糖沉淀LFP1;上清液真空浓缩到多糖浓度为2.0%左右,加入95%乙醇调节体系的乙醇浓度到50%,4℃下醇沉12小时,得粗多糖沉淀LFP2;按相同方法,加入95%乙醇调节体系的乙醇浓度到80%,得粗多糖沉淀LFP3。另外采用一次性沉降法,用80%乙醇沉降可得多糖LFP4。四种荔枝果肉多糖LFP1、LFP2、LFP3、LFP4的沉降得率分别为6.4%、53.3%、37.4%和95%。
采用DEAE-52对荔枝果肉多糖组分LFP_2进行分离,得到三个级分:LFPC_1、LFPC_2和LFPC_3;选取多糖量较高的级分LFPC_2和LFPC_3,用Sephadex G-100凝胶柱进一步分离纯化,LFPC_2过柱后分出两个级分:LFPC_2a和LFPC_2b,后者多糖量较少;LFPC_3过G-100凝胶柱后,只有一个级分。选取两个多糖级分:LFPC_2a和LFPC_3,用纸层析和凝胶层析法对LFPC_2a和LFPC_3进行纯度鉴定,证明这两个级分比较均一。
气相色谱等方法测定多糖组成,结果显示:LFPC_2a级分主要由阿拉伯糖、鼠李糖和葡萄糖组成,摩尔比为:1.00:1.37:1.21;经红外光谱,结合高碘酸氧化及Smith降解分析,显示多糖LFPC_2a存在1→2键、1→3键和1→6键,不存在1→4键,且1→3键、1→2键、1→6键之比约为:63.84:30.16:6.0。渗透凝胶色谱测定LFPC_2a的峰位分子量为43927 Da。多糖LFPC_2a粒度分布测定结果显示,不同pH条件下多糖聚集体大小顺序分别为:酸性>中性>碱性,粒度分布测定结果还表明,无论是何种条件,荔枝多糖在溶液中分布都是不均一的。LFPC_2a的X-Rays衍射分析结果显示荔枝多糖中含结晶性的多糖极少,结晶性很差,为无定形物质。扫描电镜研究发现,荔枝果肉多糖LFPC_2a在放大倍数较高的情况下,图片显示多糖表面紧密,较平整,这说明荔枝多糖分子间相互作用较强,结合紧密。热力学分析表明,与在氮气中相比,多糖LFPC_2a在空气中的热重变化规律与其相似,但变化更剧烈、更不稳定。
LFPC_3级分主要由阿拉伯糖、鼠李糖,少量半乳糖和葡萄糖组成,摩尔比为:2.42:3.44:1.00:1.65。根据洗脱曲线、茚三酮反应和紫外扫描结果,LFPC_3级分中存在少量蛋白质,LFPC_3可能是一种糖-蛋白结合物,经自动氨基酸分析仪分析显示LFPC_3由17种氨基酸组成,氨基酸总量为2626.87mg/100g。渗透凝胶色谱测定LFPC_3峰位分子量为33723 Da。
通过体外抗氧化实验,研究4种荔枝多糖LFP1、LFP2、LFP3、LFP4的抗氧化活性,结果表明:4种多糖组分均能清除DPPH自由基、超氧阴离子自由基、羟基自由基,且呈现一定的量效关系;各级分表现出很好的铁离子螯合能力,各组分的还原力随着多糖浓度的增加而提高。在各级分中LFP2抗氧化活性较好,所以选LFP2进行动物实验。通过CCl_4和酒精肝损伤小鼠模型,探讨荔枝多糖LFP2的抗氧化护肝作用,结果表明在CCl_4诱导急性肝损伤实验中,LFP2可以提高肝脏和血清中的SOD活性,并降低MDA含量,且呈一定的量效关系;同时LFP2可以提高小鼠肝脏中CAT和GSH-Px活性,从而提高肝脏的抗氧化能力,CCl_4模型组的小鼠血清中ALT、AST活性升高非常明显,LFP2中、高剂量给药组酶抑制率与阳性对照组相接近。在酒精诱导急性肝损伤实验中,荔枝多糖LFP2三个剂量组ALT、AST活性与模型组相比差异都达到了极显著水平(P<0.01),荔枝多糖中、高剂量给药组酶抑制率与阳性对照组相接近,说明荔枝多糖对酒精肝损伤有一定的保护作用。
Polysaccharide is widely found in plants, animals and microorganisms. Polysaccharides have functions of modulating immunity, reducing blood sugar and blood fat, antioxidation, anti-tumor, antivirus et al. Litchi (Litchi chinensis Sonn ) originated from China, called‘king of fruit’in South China, is one of four famous fruits in Guangdong province. Little research has been reported on polysaccharide of litchi pulp in relation to its biological activity. To further develop litchi fruits processing, studies were carried on isolation and purification of polysacchride from pulp tissue of litchi, and its antioxidant activity on liver protection was also estimated.
Twenty-one litchi cultivars were choosen in this project. The water-soluble polysaccharide content was determined. The results showed that Heiye had the higest polysaccharide content with the value of 15.72g/kg. Thus, Heiye was selected for the following trails.
Five different desication methods were used to produce litchi nuts. The results showed that the polysaccharide content and antioxidant activity of litchi polysaccharide were steady. In this project, we choose oven-drying to produce litchi nuts.
The extraction methods of litchi were systematacially studied,the methods of leaching with hot water, microwave-assisted extraction and ultrasonic-assisted extraction were investigated, respectly. The results showed that: the extraction yield of litchi extracted by hot water was 24.22%, the extraction yield of extracted by microwave-assisted was 24.26%, the extraction yield of extracted by ultrasonic-assisted was 24.32%. The extraction time of three extraction methods was different: hot water> ultrasonic-assisted> microwave-assisted. Three extraction methods had no effect on antioxidant activity of litchi polysaccharide. In this project, microwave-assisted extraction was choosen to extract the litchi polysaccharide.
Precipitation of litchi polysaccharides with different ethanol concentration: LFP1 was obtained by precipitation extracts with 20% ethanol solution. The supernatant was added ethanol to concentration of 50% to precipitate LFP2. Then the supernatant was added ethanol to concentration of 80% to precipitate LFP3. LFP4 was obtained by precipitation extracts with 80% of ethanol solution directly. Then, LFP1, LFP2, LFP3 and LFP4 were gained after purification.
LFP2 was chromatographed on a DEAE-52 anion-exchanging column to yield two major fractions:LFPC2 and LFPC3. LFPC2 and LFPC3 were subjected to further purification on a Sephadex G-100 gel filtration column. Two major fractions, LFPC_2a and LFPC_3 were collected.
By analysis of gel permeation chromatography (GPC) and gas chromatography (GC), LFPC_2a, which had a average molecular weight of 43927 Da, composed of arabinose, ribose, galactose and glucose in the molar ratio of 1.67:2.15:1.54:1.00.The results of periodate oxidation and Smith degradation showed that the molar percentage ratio of (1→3)-glycosidic linkages, (1→2)-glycosidic linkages and (1→6)-glycosidic linkages was about 63.84%:30.16%:6.0%. By using the LSCM to study LFPC_2a behavior in the solution, the conformation could be changed by the pH value. By SEM, LFPC_2a was observed the smooth figure under high-powered microscope. Thermal analysis of LFPC_2a indicated that litchi polysaccharide had similar endothermic peaks in nitrogen and air.
LFPC_3, which had a average molecular weight of 33723 Da, composed of arabinose, rhamnose, galactose and glucose in the molar ratio of 2.42:3.44:1.00:1.65. The polysaccharide absorption peak showed similar time and shape as the protein absorption peak, suggesting that LFP3 is likely a glycoprotein.
In vitro antioxidant activity of LFP1, LFP_2, LFP3 and LFP4 showed that exhibited a dose-dependent free radical scavenging activity against DPPH radical, superoxide anions, hydroxyl radical, chelating ability and reducing power. Among the four different fractions, LFP_2 showed the strongest scavenging activity.
The antioxidant activity of litchi polysaccharide LFP_2 for mouse liver protection was investigated by CC14-induced and ethanol-induced liver injury experiments. Results indicated that LFP_2 increased SOD activity of mouse liver and serum, and reduced the content of MDA by dose-dependent pattern. LFP_2 also increased mouse liver CAT and GSH-Px activities. In the experiment of CC14-induced mouse oxidative injury, ALT and AST activities of mouse liver increased significantly, and high and medium dossage LFP_2 could inhibit the enzyme activities of ALT and AST. Results of experiment of ethanol-induced mouse oxidative injury showed that, three dossage of LFP_2 could also inhibit the enzyme activities of ALT and AST. These results indicated that litchi polysaccharide could increase the activity of antioxidant enzymatic system and antioxidant ability of liver to protect from injury.
引文
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