荔枝果肉多糖超声微波酶解协同提取及其生物活性初析
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摘要
荔枝是华南地区特色水果,其果香独特、营养丰富,具有显著的滋补功效。现代研究表明,荔枝果肉具有抗氧化、抗肿瘤、增强免疫等活性作用,而荔枝果肉活性多糖被证实是其具有保健作用的重要物质基础之一。本文通过优化荔枝果肉多糖的超声微波酶解协同提取工艺,比较了此方法与传统热水法提取的荔枝多糖的体外抗氧化活性、免疫调节活性及其结构特征差异；确定荔枝多糖离子层析分级条件,比较其组分体外抗氧化活性、免疫调节活性,并初步解析其基本结构,为荔枝制品尤其为功能食品的开发及应用奠定基础。论文的主要研究结果如下：
1.超声微波酶解协同提取荔枝多糖：采用响应面法分析设计五因素三水平试验,优化荔枝果肉多糖的超声微波酶解协同提取工艺。经分析并结合验证试验,确定荔枝多糖的最佳提取工艺条件为：纤维素酶(酶活为30U/mg)添加量14mg/g(与底物之比1,料液比1：10,温度49℃,pH值4.8,提取时间12min。在该条件下,荔枝多糖得率可达23.31%,比传统热水法、超声法、微波法分别提高18.95%、4.37%和17.10%(P<0.05)。
2.超声微波酶解协同法与热水法提取荔枝多糖的活性比较及结构初析：比较两种方法提取荔枝多糖(LCP)体外还原Fe3+能力、清除DPPH自由基、ABTS自由基的结果显示,超声微波酶解法提取的LCP清除DPPH·和ABTS·+的IC50分别为427μg/mL、203μg/mL,显著低于同浓度下热水法提取的LCP清除DPPH·(?)和ABTS·+的IC50(2734μg/mL、1590μg/mL, P<0.05); FRAP抗氧化值为0.32μmol/mg,显著高于热水法提取LCP的FRAP值(0.16μmol/mg, P<0.01),表明超声微波酶解法提取LCP的抗氧化能力显著高于热水法提取的LCP。同时,细胞活性实验的结果表明,超声微波酶解法提取的LCP促进脾淋巴细胞增殖和NK细胞杀伤能力的活性显著(P<0.05)高于热水法提取的LCP。
对两种方法提取的LCP特性分析的结果表明,超声微波酶法提取的LCP水溶性多糖含量为57.26%,糖醛酸含量为12.39%；热水法提取的LCP水溶性多糖的含量47.62%,糖醛酸的含量23.69%；红外光谱、气相色谱—质谱(GC-MS)、凝胶色谱的分析结果显示,超声微波酶法提取的LCP为具卤素取代的α-吡喃型多糖,由木糖、鼠李糖、核糖、甘露糖、葡萄糖、半乳糖和阿拉伯糖等单糖组成(摩尔比为1：4.39：6.05：16.07：65.20：23.62：15.25),分子量为7.92x104和9.09×10。；热水法提取的LCP为具O-糖苷键结合的有蛋白的多糖,由木糖、鼠李糖、核糖、甘露糖、葡萄糖、半乳糖和阿拉伯糖等单糖组成(摩尔比为1：2.59：5.77：8.13：22.49：22.50：26.02),分子量为6.01×104。两种方法提取LCP的水溶性多糖含量、单糖组成和分子量的差异可能是造成其活性差异的基础。
3.层析条件优化及级分分离：优化荔枝多糖DEAE-52离子层析的分级条件,确定其最适分级条件为：上样浓度为4.2mg/ml,上样体积为8ml,柱流速为1ml/min)径长比为1：30,上样重复利用两次,梯度洗脱液为0.1mol/LNaCl,0.2mol/LNaCl,0.3 mol/LNaCl和0.3mol/LNaOH。采用该方法对提取的荔枝粗多糖进行分级纯化,得到LCP1-5五个组分,洗脱率为91.10%：0.78%：0.42%：0.11%：1.09%。
4.荔枝多糖主级分的生物活性及其结构特征比较：选取DEAE-52层析分离纯化得到的主组分LCP1和LCP5,比较其FRAP抗氧化活性及促进脾淋巴细胞增殖、NK细胞杀伤的能力。结果显示,LCP5的FRAP抗氧化活性(1.28μmol/mg)显著高于LCP1(0.136μmol/mg, P<0.05)。同时,LCP5促进脾淋巴细胞增殖活性显著强于LCP1(P<0.01)；在浓度为300μg/mL和400μg/mL(?)时,其促进NK细胞杀伤能力的活性显著高于LCP1(P<0.05)。对其理化特性分析的结果显示,LCP1的水溶性多糖含量为63.16%,糖醛酸含量为32.36%,紫外光谱分析显示其含有少量核酸和蛋白质；LCP5的水溶性多糖含量为42.84%,糖醛酸含量为10.82%。红外光谱、GC-MS和高效凝胶渗透色谱的分析结果显示,LCP1为具端炔基酸性多糖,由核糖、鼠李糖、甘露糖、葡萄糖、阿拉伯糖、半乳糖按1：1.58：2.66：4.97：9.15：11.14构成,其数均分子量为5.45×104；LCP5为具卤素取代的α-吡喃型酸性多糖,由核糖、鼠李糖、甘露糖、葡萄糖、阿拉伯糖、半乳糖按1：0.75：1.49：3.20：4.82：2.09构成,其数均分子量为1.41×104。
Litchi is delicious and nutritiou, is saw as "the treasure of the fruit", all parts of which could be used as medicine. Studies have shown that litchi could improve antioxidant、antitumor and immunomodulatory effect and clearly pointed out that litchi polysaccharides (LCP) is one of the main functional active components. In this paper, litchi pulp was used as raw materials, the optimum conditions of ultrasonic-microwave-enzyme -assisted extraction technology for LCP were determined; column chromatography on DEAE-52 cellulose was used to isolate and purify the major polysaccharides extracted from litchi pulp; the antioxidant、immunomodulatory effects and strucure of crude LCP and major purify LCP were compared. The study results will offer a new clue for the deep processing of LCP, as well as provide theoretical foundation and technical guidance for the development of healthy products. The main contents and results are shown as below:
1. Optimization of extraction condition of LCP:The process of using ultrasonic-microwave-enzyme synergistic method to extract polysaccharides from litchi pulp was optimized through the response surface methodology of Box-Benhnek central combination experimental design. The regression model of five factors for the extraction of litchi polysaccharides, which were the amount of enzyme addition, solid-liquid ration, temperature and time was established. Through the experiments and analysis, the optimal process conditions to extract litchi polysaccharides were as follows:the amount that cellulose added wasl4mg/g (enzyme activity was 30U/mg), the solid-liquid ratio was 1: 10, the temperature was set at 49℃, the pH value was 4.8 and the time used wasl2min. From those conditions, the yield rate of polysaccharides extraction was 23.3%, which were 19.0%,4.4% and 17.1% (P<0.05) higher than those were extracted using the method of traditional hot water, ultrasonic and microwave respectively.
2. Comparison of functional activity and structure of LCP from ultrasonic-microwave- enzyme -assisted extraction technology and hot water:The antioxidant activity of extract from Litchi was assayed by ferric reducing (FRAP) assay, DPPH and ABTS radical scavenging and the reslutes from ultrasonic- microwave-enzyme -assisted extraction technology showed that FRAP value of LCP from ultrasonic- microwave-enzyme -assisted extraction technology was 0.32μmol FeSO4/mg, higher than hot water(0.16μmol FeSO4/mg), the positive correlation (P<0.01) existed between reductive capacity and concentration(P<0.01); IC50 value of DPPH and ABTS radical scavenging activity was respectively 427μg/mL、203μg/mL, lower than hot water (DPPH and ABTS radical scavenging activity was respectively 2734μg/mL and 1590μg/mL). At the same time, the effect from ultrasonic -microwave -enzyme -assisted extraction technology was better than hot water to increase the cytotoxic activity of NK cells(P<0.05) and promote ConA-induced proliferation of the spleen lymphocyte (P<0.01).
Analysis results of physiochemical properties showed that the total sugar content of LCP from ultrasonic -microwave -enzyme -assisted extraction technology was 57.26%, the aldonic acid content was 12.39%; the total sugar content of LCP from hot water was 47.62%, the aldonic acid content was 23.69%. Analysis results of HPGPC、GC-MS、IR and UV showed that LCP from ultrasonic -microwave -enzyme -assisted extraction technology was polysaccharides includingα-pyrans and haligen, composed of xylose、rhamnose、ribise、mannose、glucose、galactose、arabinose, and the molar ratio was 1: 4.39:6.05:16.07:65.20:23.62:15.25, its relative molecular weight was 7.92×104、9.09×103; LCP from hot water was protein-polysaccharides with O-glucosides and protein, composed of xylose、rhamnose、ribise、mannose、glucose、galactose、arabinose, and the molar ratio was 1:2.59:5.77:8.13:22.49:22.50:26.02, its relative molecular weight was 6.01×104.
3.Isolation and purification:The DEAE-52 ionic exchange chromatographic method was appied to purify the rude polysaccharide, and five components obtained and the ratio was 91.10%:0.78%:0.42%:0.11%:1.09%.The optional isolation conditions were established as:the column loading:4.2mg/mL; the column volume:8mL; the elution speed:1mL/min; the ratio of diameter and length:30:1; operate twice.
4.The analysis of active components of LCP:The antioxidant activity of LCP1 and LCP5 was studied by FRAP, and the immunomodulatory effect was evaluated by using normal mice immunocytes with respect to lymphocytes proliferation and NK activity. The results indicated that the FRAP of LCP5(1.281μmol/mg) was higher than LCP1(0.136μmol/mg, P<0.05). LCP5 was better than LCP1(P<0.05) to increase the cytotoxic activity of NK cells and at the dose of 300～400μg/mL, LCP5 was better than LCP1(P<0.05) to promote ConA- induced proliferation of the spleen lymphocyte (P<0.05).
Analysis results of physiochemical properties showed that the total sugar content of LCP1 was 63.16%, the aldonic acid content was 32.36%; the total sugar content of LCP5 was42.84%, the aldonic acid content was 10.82%. Analysis results of HPGPC、GC-MS、IR and UV showed that LCP was acid polysaccharides with acetylene series, composed of ribise、rhamnose、mannose、glucose、arabinose、galactose and the molar ratio was 1: 1.58:2.66:4.97:9.15:11.14, its relative molecular weight was 5.45×104; LCP5 was acid polysaccharides with halogen, composed of ribise、rhamnose、mannose、glucose、 arabinose、galactose and the molar ratio was 1:0.75:1.49:3.20:4.82:2.09, its relative molecular weight was 1.41×104.
1.超声微波酶解协同提取荔枝多糖：采用响应面法分析设计五因素三水平试验,优化荔枝果肉多糖的超声微波酶解协同提取工艺。经分析并结合验证试验,确定荔枝多糖的最佳提取工艺条件为：纤维素酶(酶活为30U/mg)添加量14mg/g(与底物之比1,料液比1：10,温度49℃,pH值4.8,提取时间12min。在该条件下,荔枝多糖得率可达23.31%,比传统热水法、超声法、微波法分别提高18.95%、4.37%和17.10%(P<0.05)。
2.超声微波酶解协同法与热水法提取荔枝多糖的活性比较及结构初析：比较两种方法提取荔枝多糖(LCP)体外还原Fe3+能力、清除DPPH自由基、ABTS自由基的结果显示,超声微波酶解法提取的LCP清除DPPH·和ABTS·+的IC50分别为427μg/mL、203μg/mL,显著低于同浓度下热水法提取的LCP清除DPPH·(?)和ABTS·+的IC50(2734μg/mL、1590μg/mL, P<0.05); FRAP抗氧化值为0.32μmol/mg,显著高于热水法提取LCP的FRAP值(0.16μmol/mg, P<0.01),表明超声微波酶解法提取LCP的抗氧化能力显著高于热水法提取的LCP。同时,细胞活性实验的结果表明,超声微波酶解法提取的LCP促进脾淋巴细胞增殖和NK细胞杀伤能力的活性显著(P<0.05)高于热水法提取的LCP。
对两种方法提取的LCP特性分析的结果表明,超声微波酶法提取的LCP水溶性多糖含量为57.26%,糖醛酸含量为12.39%；热水法提取的LCP水溶性多糖的含量47.62%,糖醛酸的含量23.69%；红外光谱、气相色谱—质谱(GC-MS)、凝胶色谱的分析结果显示,超声微波酶法提取的LCP为具卤素取代的α-吡喃型多糖,由木糖、鼠李糖、核糖、甘露糖、葡萄糖、半乳糖和阿拉伯糖等单糖组成(摩尔比为1：4.39：6.05：16.07：65.20：23.62：15.25),分子量为7.92x104和9.09×10。；热水法提取的LCP为具O-糖苷键结合的有蛋白的多糖,由木糖、鼠李糖、核糖、甘露糖、葡萄糖、半乳糖和阿拉伯糖等单糖组成(摩尔比为1：2.59：5.77：8.13：22.49：22.50：26.02),分子量为6.01×104。两种方法提取LCP的水溶性多糖含量、单糖组成和分子量的差异可能是造成其活性差异的基础。
3.层析条件优化及级分分离：优化荔枝多糖DEAE-52离子层析的分级条件,确定其最适分级条件为：上样浓度为4.2mg/ml,上样体积为8ml,柱流速为1ml/min)径长比为1：30,上样重复利用两次,梯度洗脱液为0.1mol/LNaCl,0.2mol/LNaCl,0.3 mol/LNaCl和0.3mol/LNaOH。采用该方法对提取的荔枝粗多糖进行分级纯化,得到LCP1-5五个组分,洗脱率为91.10%：0.78%：0.42%：0.11%：1.09%。
4.荔枝多糖主级分的生物活性及其结构特征比较：选取DEAE-52层析分离纯化得到的主组分LCP1和LCP5,比较其FRAP抗氧化活性及促进脾淋巴细胞增殖、NK细胞杀伤的能力。结果显示,LCP5的FRAP抗氧化活性(1.28μmol/mg)显著高于LCP1(0.136μmol/mg, P<0.05)。同时,LCP5促进脾淋巴细胞增殖活性显著强于LCP1(P<0.01)；在浓度为300μg/mL和400μg/mL(?)时,其促进NK细胞杀伤能力的活性显著高于LCP1(P<0.05)。对其理化特性分析的结果显示,LCP1的水溶性多糖含量为63.16%,糖醛酸含量为32.36%,紫外光谱分析显示其含有少量核酸和蛋白质；LCP5的水溶性多糖含量为42.84%,糖醛酸含量为10.82%。红外光谱、GC-MS和高效凝胶渗透色谱的分析结果显示,LCP1为具端炔基酸性多糖,由核糖、鼠李糖、甘露糖、葡萄糖、阿拉伯糖、半乳糖按1：1.58：2.66：4.97：9.15：11.14构成,其数均分子量为5.45×104；LCP5为具卤素取代的α-吡喃型酸性多糖,由核糖、鼠李糖、甘露糖、葡萄糖、阿拉伯糖、半乳糖按1：0.75：1.49：3.20：4.82：2.09构成,其数均分子量为1.41×104。
Litchi is delicious and nutritiou, is saw as "the treasure of the fruit", all parts of which could be used as medicine. Studies have shown that litchi could improve antioxidant、antitumor and immunomodulatory effect and clearly pointed out that litchi polysaccharides (LCP) is one of the main functional active components. In this paper, litchi pulp was used as raw materials, the optimum conditions of ultrasonic-microwave-enzyme -assisted extraction technology for LCP were determined; column chromatography on DEAE-52 cellulose was used to isolate and purify the major polysaccharides extracted from litchi pulp; the antioxidant、immunomodulatory effects and strucure of crude LCP and major purify LCP were compared. The study results will offer a new clue for the deep processing of LCP, as well as provide theoretical foundation and technical guidance for the development of healthy products. The main contents and results are shown as below:
1. Optimization of extraction condition of LCP:The process of using ultrasonic-microwave-enzyme synergistic method to extract polysaccharides from litchi pulp was optimized through the response surface methodology of Box-Benhnek central combination experimental design. The regression model of five factors for the extraction of litchi polysaccharides, which were the amount of enzyme addition, solid-liquid ration, temperature and time was established. Through the experiments and analysis, the optimal process conditions to extract litchi polysaccharides were as follows:the amount that cellulose added wasl4mg/g (enzyme activity was 30U/mg), the solid-liquid ratio was 1: 10, the temperature was set at 49℃, the pH value was 4.8 and the time used wasl2min. From those conditions, the yield rate of polysaccharides extraction was 23.3%, which were 19.0%,4.4% and 17.1% (P<0.05) higher than those were extracted using the method of traditional hot water, ultrasonic and microwave respectively.
2. Comparison of functional activity and structure of LCP from ultrasonic-microwave- enzyme -assisted extraction technology and hot water:The antioxidant activity of extract from Litchi was assayed by ferric reducing (FRAP) assay, DPPH and ABTS radical scavenging and the reslutes from ultrasonic- microwave-enzyme -assisted extraction technology showed that FRAP value of LCP from ultrasonic- microwave-enzyme -assisted extraction technology was 0.32μmol FeSO4/mg, higher than hot water(0.16μmol FeSO4/mg), the positive correlation (P<0.01) existed between reductive capacity and concentration(P<0.01); IC50 value of DPPH and ABTS radical scavenging activity was respectively 427μg/mL、203μg/mL, lower than hot water (DPPH and ABTS radical scavenging activity was respectively 2734μg/mL and 1590μg/mL). At the same time, the effect from ultrasonic -microwave -enzyme -assisted extraction technology was better than hot water to increase the cytotoxic activity of NK cells(P<0.05) and promote ConA-induced proliferation of the spleen lymphocyte (P<0.01).
Analysis results of physiochemical properties showed that the total sugar content of LCP from ultrasonic -microwave -enzyme -assisted extraction technology was 57.26%, the aldonic acid content was 12.39%; the total sugar content of LCP from hot water was 47.62%, the aldonic acid content was 23.69%. Analysis results of HPGPC、GC-MS、IR and UV showed that LCP from ultrasonic -microwave -enzyme -assisted extraction technology was polysaccharides includingα-pyrans and haligen, composed of xylose、rhamnose、ribise、mannose、glucose、galactose、arabinose, and the molar ratio was 1: 4.39:6.05:16.07:65.20:23.62:15.25, its relative molecular weight was 7.92×104、9.09×103; LCP from hot water was protein-polysaccharides with O-glucosides and protein, composed of xylose、rhamnose、ribise、mannose、glucose、galactose、arabinose, and the molar ratio was 1:2.59:5.77:8.13:22.49:22.50:26.02, its relative molecular weight was 6.01×104.
3.Isolation and purification:The DEAE-52 ionic exchange chromatographic method was appied to purify the rude polysaccharide, and five components obtained and the ratio was 91.10%:0.78%:0.42%:0.11%:1.09%.The optional isolation conditions were established as:the column loading:4.2mg/mL; the column volume:8mL; the elution speed:1mL/min; the ratio of diameter and length:30:1; operate twice.
4.The analysis of active components of LCP:The antioxidant activity of LCP1 and LCP5 was studied by FRAP, and the immunomodulatory effect was evaluated by using normal mice immunocytes with respect to lymphocytes proliferation and NK activity. The results indicated that the FRAP of LCP5(1.281μmol/mg) was higher than LCP1(0.136μmol/mg, P<0.05). LCP5 was better than LCP1(P<0.05) to increase the cytotoxic activity of NK cells and at the dose of 300～400μg/mL, LCP5 was better than LCP1(P<0.05) to promote ConA- induced proliferation of the spleen lymphocyte (P<0.05).
Analysis results of physiochemical properties showed that the total sugar content of LCP1 was 63.16%, the aldonic acid content was 32.36%; the total sugar content of LCP5 was42.84%, the aldonic acid content was 10.82%. Analysis results of HPGPC、GC-MS、IR and UV showed that LCP was acid polysaccharides with acetylene series, composed of ribise、rhamnose、mannose、glucose、arabinose、galactose and the molar ratio was 1: 1.58:2.66:4.97:9.15:11.14, its relative molecular weight was 5.45×104; LCP5 was acid polysaccharides with halogen, composed of ribise、rhamnose、mannose、glucose、 arabinose、galactose and the molar ratio was 1:0.75:1.49:3.20:4.82:2.09, its relative molecular weight was 1.41×104.
引文
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