【作者】 常清泉；

【导师】 陆娟； 陈瑞战；

【作者基本信息】 长春师范大学 ， 化学， 2018， 硕士

【摘要】 长白山产软枣猕猴桃（Actinidia arguta）和狗枣猕猴桃（Actinidia kolomikta）同属植物系猕猴桃科猕猴桃属多年生落叶藤本植物,在我国尤其是东北地区分布较广,目前大都处于野生状态未被广泛利用,开发潜力较大。本文以软枣猕猴桃、狗枣猕猴桃茎叶为原料,对猕猴桃属植物茎叶多糖的提取、分离、纯化、结构及抗氧化活性等方面进行了较为系统的研究。得到结论如下:1.多糖的提取工艺研究以粗多糖提取得率为指标,利用不同提取方法分别提取软枣猕猴桃茎多糖,软枣猕猴桃叶多糖、狗枣猕猴桃叶多糖,以筛选合适的提取方法。利用单因素试验和响应面优化其相应的提取工艺。（1）软枣猕猴桃茎粗多糖微波提取的最优工艺为:微波时间40.03 min,微波功率314.63 W,液料比59.02 mL/g。在此条件下,软枣猕猴桃茎粗多糖的提取得率为2.90%。（2）软枣猕猴桃叶粗多糖的超声提取最优条件为:提取时间41.55 min,超声波功率928.8W,液料比38.62 mL/g,在此条件下,软枣猕猴桃叶多糖提取得率理论值为3.21%。（3）狗枣猕猴桃叶粗多糖闪式提取的最佳条件为:液料比17.83 mL/g、闪式提取的转速为9500 r/min、提取温度43.36℃,提取时间10.27 min,在此条件下,狗枣猕猴桃叶粗多糖得率理论值为6.40%。2.多糖分离纯化及结构初步分析分别利用不同规格的琼脂糖凝胶和葡聚糖凝胶柱色谱对软枣猕猴桃茎多糖（AAS）、软枣猕猴桃叶多糖（AAL）、狗枣猕猴桃茎多糖（AKS）和狗枣猕猴桃叶多糖（AKL）进行分离纯化,得到AAS-1-1、AAL-1-1、AAL-2-1、AAL-3-1、AKS-1-1、AKS-2-1、AKL-1-1、AKL-2-1八个均一多糖组分。八个均一多糖的总糖含量分别为88.78%（AAS-1-1）、80.46%（AAL-1-1）、89.86%（AAL-2-1）、85.78%（AAL-3-1）、80.45%（AKS-1-1）、82.66%（AKS-2-1）、81.26%（AKL-1-1）、90.23%（AKL-2-1）。糖醛酸含量分别为16.71%（AAS-1-1）、18.74%（AAL-1-1）、1.07%（AAL-2-1）、0.65%（AAL-3-1）、6.21%（AKS-1-1）、1.02%（AKS-2-1）、22.45%（AKL-1-1）、6.65%（AKL-2-1）。蛋白质含量相对较低。利用高效凝胶色谱法（HPGPC）测定了八个均一多糖的分子量,分别为103722（AAS-1-1）、109629.8（AAL-1-1）、62715（AAL-2-1）、62381（AAL-3-1）、125314.6（AKS-1-1）、65574.58（AKS-2-1）、354813.5（AKL-1-1）、344839.7（AKL-2-1）Da。红外光谱显示八个均一多糖均具有多糖类物质特征吸收峰。利用PMP柱前衍生-高效液相色谱法（HPLC）对八个均一多糖的结构进行了初步分析。八个均一多糖均是由鼠李糖（Rha）、葡萄糖醛酸（GlcA）、半乳糖（Gal）、葡萄糖（Glc）、半乳糖醛酸（GalA）、木糖（Xyl）、阿拉伯糖（Ara）等七种单糖以不同的摩尔比组成。3.多糖抗氧化活性研究考察了四种粗多糖及八个均一多糖对1,1-二苯基-2-苦肼基（DPPH·）、羟基自由基（·OH）清除能力并对比了八个均一多糖的抗氧化活性。所测多糖对上述两种自由基均有抗氧化活性,活性能力与多糖的浓度呈现正比关系。利用清除自由基的半数浓度(IC₅₀)值比较其抗氧化活性大小,八个均一多糖清除DPPH·能力的顺序为:AAL-2-1（2.93 mg/mL）>AAL-1-1（3.06 mg/mL）>AAS-1-1（3.13 mg/mL）>AKS-1-1（3.39 mg/mL）>AKS-2-1（3.40 mg/mL）=AKL-1-1（3.40 mg/mL）>AKL-2-1（3.45 mg/mL）>AAL-3-1（3.70 mg/mL）。八个均一多糖清除·OH的顺序为:AAL-2-1（2.51 mg/mL）>AAL-1-1（2.57 mg/mL）>AAS-1-1（2.99 mg/mL）>AAL-3-1（3.16 mg/mL）>AKS-1-1（3.56 mg/mL）>AKS-2-1（3.59 mg/mL）>AKL-1-1（3.90 mg/mL）>AKL-2-1（4.00 mg/mL）。均一多糖的抗氧化活性与多糖的分子量、糖醛酸含量有直接关系,分子量越小、糖醛酸含量越高,抗氧化活性越高。分离得到的八个均一多糖可以探索作为天然抗氧化剂应用功能性食品和药品。更多还原

【Abstract】 Actinidia arguta(Sieb.et Zucc.)Planch and Actinidia kolomikta(Rupr.et Maxim.)Planch were belong to Actinidiaceae and mainly distributed in the ChangBai Mountain.In this study,the polysaccharides from stems and leaves of Actinidia arguta and Actinidia kolomikta were comprehensively investigated,including extraction,purification,characterization of polysaccharides and antioxidant activities.The results were as follows:1.The extraction tecnology of polysaccharidesWith the yields of crude polysaccharide as the index,different extraction methods were used to extract the polysaccharides from the stems and leaves of Actinidia arguta and Actinidia kolomikta to select the the appropriate extraction method.Single test and Response Surface Methodology was used to optimize the extraction tecnology of polysaccharides from the stems and leaves.And the results are as follows:(1)The optimum condition of microwave-assisted extraction to extract the polysaccharides form the stems of Actinidia arguta were microwave time 40.03 min,microwave power 314.64 W,water to material ratio 59.02 mL/g,the predicted optimal yield of AAS was 2.90%.(2)The optimum condition of ultrasonic tecnology to extact the polysaccharides form the leaves of Actinidia arguta were ultrasonic time 40.00 min,liquid-solid ratio 40 mL/g and the ultrasonic power 900 W.Under the condition the yield of leaves polysaccharide was 4.50%.(3)The optimum condition of ultrasonic tecnology to extact the polysaccharides form the leaves of Actinidia kolomikta was that liquid-solid ratio,T10 basic speed extraction temperature,and extraction time were 17.38 mL/g,9500 r/min,43.36℃,10.27 min,respectively and the extraction yield was 6.40 %.2.Isolation,purification and preliminary characterization of polysaccharidesThe polysaccharides were applied to a DEAE Sepharose fast flow anion-exchange chromatography and Sephadex G-100 chromatography,using water and sodium chloride at different concentrations as eluent respectively to give eight polysaccharide fractions,named AAS-1-1,AAL-1-1,AAL-2-1,AAL-3-1,AKS-1-1 AKS-2-1,AKL-1-1 and AKL-2-1.The total polysaccharide contents of eight polysaccharides were 88.78%(AAS-1-1),80.46%(AAL-1-1),89.86%(AAL-2-1),85.78%(AAL-3-1),80.45%(AKS-1-1),82.66%(AKS-2-1),81.26%(AKL-2-1),90.23%(AKL-2-1)respectively.The contents of uronic acid of eight polysaccharides were 16.71%(AAS-1-1),18.74%(AAL-1-1),1.07%(AAL-2-1),0.65%(AAL-3-1),6.21%(AKS-1-1),1.02%(AKS-2-1),22.45%(AKL-2-1),6.65%(AKL-2-1),respectively.The protein contents were relatively low.The molecular weight of AAS-1-1,AAL-1-1,AAL-2-1,AAL-3-1,AKS-1-1,AKS-2-1,AKL-1-1 and AKL-2-1 were determined by high performance gel permeation chromatography,and the results were as follows: 103722(AAS-1-1),109629.8(AAL-1-1),62715(AAL-2-1),62381(AAL-3-1),125314.6(AKS-1-1),65574.58(AKS-2-1),354813.5(AKL-1-1),344839.7(AKL-2-1)Da,respectively.Infrared spectrum scanning results showed that AAS-1-1,AAL-1-1,AAL-2-1,AAL-3-1,AKS-1-1,AKS-2-1,AKL-1-1 and AKL-2-1 had characteristic absorption peak of polysaccharides.The monosaccharide compositions of polysaccharides were analyzed by HPLC.All the eight polysaccharides were composed of rhamnose(Rha),glucuronic acid(GlcA),galactose(Gal),glucose(Glu),galacturonic acid(GalA),xylose(Xyl)and arabinose(Ara)at different molar ratio.3.The antioxidant activity of polysaccharidesThe antioxidant activities of AAS,AAL,AKS,AKL,AAS-1-1,AAL-1-1,AAL-2-1,AAL-3-1,AKS-1-1,AKS-2-1,AKL-1-1 and AKL-2-1 were estimated by measuring the scavenging activities on hydroxyl free radical(?OH)and DPPH·.And the results showed that all the polysaccharides have the abilities of scavenging DPPH? and ?OH.And the scavenging activities were dependent on the mass concentration of polysaccharide samples.According to the concentration inhibiting 50% radical,the order of scavenging ability on ?DPPH by eight homogeneous polysaccharides is as follows: AAL-2-1 > AAL-1-1 > AAS-1-1 > AKS-1-1 > AKS-2-1 = AKL-1-1 > AKL-2-1 > AAL-3-1.The order of scavenging ability on ?OH by eight homogeneous polysaccharides is as follows: AAL-1-1 > AAS-1-1 > AAL-2-1 > AAL-3-1 > AKS-1-1 > AKS-2-1 > AKL-1-1 > AKL-2-1.These results showed that the antioxidant activity was directly related to the molecular weight of polysaccharides,the concentration of the uronic acid,the smaller the molecular weight,the higher the concentration of the uronic acid acid,the higher the antioxidant activity.It is shown that these polysaccharides have significant antioxidant activities and can be explored as a natural antioxidant for food and medicine.更多还原