【作者】 张亮亮；

【导师】 林鹏； 林益明；

【作者基本信息】 厦门大学 ， 植物学， 2009， 博士

【摘要】 对植物单宁化学结构进行快速、准确的分析测定是筛选和开发有重大应用价值的植物单宁资源的前提。本文利用现代仪器分析技术对我国南方几种经济植物单宁的化学结构和抗氧化能力进行了系统研究，主要研究内容及结果有：1．首次利用高效液相色谱-二极管阵列检测法（HPLC-DAD）研究了不同反应条件对木榄花萼原花色素降解（正丁醇／HCI法）产物花青定及反应副产物的影响。经HPLC-DAD检测分析发现反应体系含水量对红树植物木榄花萼原花色素降解产物及副产物影响较大。当反应体系含水量在5％～15％的条件下，转化产物随着反应体系含水量的增加，花青定的转化率也随之增加，副产物的转化率却随之减少，总转化率（即花青定加上副产物的总转化量）呈增加趋势；在反应体系含水量15％以上，随含水量的增加，转化产物含量呈现下降的趋势；增加反应体系中原花色素样品含量并没有明显增加转化产物的转化率，副产物的转化率相对稳定；不同反应时间处理得到的转化产物经HPLC-DAD检测分析并未发现转化产物花青定和副产物表现出明显的变化规律，总转化率相对稳定。研究表明4PLC-DAD技术能够准确地分析测定植物样品中原花色素含量及原花色素结构单元组成类型。2．利用基质辅助激光解析电离飞行时间（MALDI-TOF）质谱分析测定了木榄花萼中原花色素的结构单元组成类型、平均聚合度和平均相对分子质量，首次报道了以木榄花萼原花色素为原料，通过正丁醇/HCI法酸解反应制备了花青定粗产品，测定了花青定粗产品对二苯基苦基肼自由基（DPPH）的清除能力及铁离子还原/抗氧化能力（FRAP）。木榄花萼中所含原花色素结构单元组成类型主要为儿茶素和表儿茶素（原花青素的结构单元）。平均聚合度为7．5，平均相对分子质量为2081．60；花青定粗产品具有很强的清除自由基能力(半抑制率浓度IC₅₀为43．89μg／L)，及较高的FRAP抗氧化能力（7．72 mmol AAE／g）。创新性地建立了一种利用自然界广泛存在的原花色素资源通过酸解转化制备花青定等花色素的新途径。3．利用MAL-DI-TOF质谱联合NMR及HPL-C分析测定了李子果肉、海南蒲桃果实及橄榄各部分中所含单宁的化学结构及其抗氧化活性。（1）李子果肉中总酚含量为82．89±13．12 mg／g，可溶缩合单宁含量为14．31±9．27 mg／g；构成李子果肉单宁的黄烷-3-醇结构单元主要是表儿茶素，在化学结构上属于原花青素类型，且大部分聚合物的结构单元之间存在A型和B型2种连接方式，平均聚合度为5．3，平均相对分子质量为1 583．7。经DPPH·法测定发现李子果肉单宁具有较高的自由基清除能力(IC₅₀为57．98μg／mL)。（2）海南蒲桃果实核中所含单宁类型为鞣花单宁，其结构为葡萄糖核、没食子酸酯、鞣花酸及其衍生物构成。海南蒲桃果实皮中所含单宁类型为缩合单宁，缩合单宁黄烷-3-醇结构单元主要为表阿福豆素通过B型连接而形成的低聚物，即在结构上属于天竺葵色素类型，MAL-DI-TOF质谱图中最多可观测到十一聚体的存在。经DPPH．和FRAP两种体外抗氧化模型检测发现，海南蒲桃果实单宁提取物具有很强的自由基清除作用和抗氧化能力。海南蒲桃果实是一种值得大力开发的天然抗氧化剂资源。（3）橄榄茎皮缩合单宁属于原花青素和原翠雀素类型，其黄烷-3-醇结构单元在空间立体结构上属于2，3-反式结构。并在原花青素和原翠雀素的结构单元表儿茶素和表棓儿茶素中发现有没食子酸酯的存在。橄榄茎皮缩合单宁平均聚合度为5．3，平均相对分子质量为1 578．25。MAL-DI-TOF质谱、HPLC及NMR技术是分析多分散的植物单宁聚合物的理想工具。经DPPH和FRAP体外抗氧化模型检测发现橄榄叶片、小枝及茎皮中所含单宁组分均表现出较强的自由基清除作用(IC₅₀：56．86，62．31和54．80μg／mL)和抗氧化能力（4．28，3．74和4．49 mmolAAE／g）。更多还原

【Abstract】 In order to screen and exploit vegetable tannins which have importantproperties in practical application,convenient and reliable methods arerequired to determine vegetable tannins structure.In the present study,wehave studied the following several aspects on the chemical structure andantioxidant activity of vegetable tannins from several plants in south China:1.The effect of different reaction conditions on cyanidin from degradation ofproanthocyanidin extracted from Bruguiera gymnorrhiza calyces and sideproducts were studied by high performance liquid chromatography-diode arraydetector （HPLC-DAD） for the first time.The cyanidin and corresponding sideproduct formed during butanol/HCI hydrolysis were separated and quantifiedby HPLC-DAD.The degradation products of proanthocyanidin were effectgreatly by water content of reaction solvents.The yield of cyanidin increasedwith the increasing the water content （from 5% to 15%） of reaction solvents,but the yield of side products declined.The yieldof cyanidin and side productsdecreased with the increasing the water content when the water content wasabove 15%.However,there is no great effect of proanthocyanidinconcentration and reaction time on the yield of cyanidin and side products.HPLC-DAD is a powerful method for the analysis of the content and structureunit of proanthocyanidin.2.Types of structural units,degree of polymerization and the meanmolecular weight of proanthocyanidin extracted from B.gymnorrhiza calyxwere characterized by matrix-assisted laser desorption/ionization time-of-flightmass spectrometry （MALDI-TOF MS）.Crude cyanidin products were preparedfrom proanthocyanidin of B.gymnorrhiza by means of butanol/HCI reaction forthe first time.In addition,the effects of crude cyanidin products on freeradical-scavenging and antioxidant activity were determined by using1,1-diphenyl-2-picryhydrazyl radical （DPPH·） scavenging activity and ferric reducing/antioxidant power （FRAP） model systems.Catechin/epicatechin（procyanidin） was the basic unit occurring in procyanidin of B.gymnorrhiza.The average degree of polymerization was 7.5,and mean molecular mass was2081.60.Crude cyanidin products showed a very good DPPH radicalscavenging activity (IC₅₀,the half-inhibition concentration was 43.89μg/L) andferric reducing/antioxidant power （7.72 mmol AAE/g）.A new method forpreparing cyanidin and anthocyanidin from easilyavailable proanthocyanidinby means of butanol/HCI reaction was established.3.Tannins from Prunus salicina,Syzygium cuminic and Canarium albumwere charactered using MALDI-TOF MS combined with NMR and HPLCanalysis for the first time.（1） The content of total phenolics and extractablecondensed tannins in P.salicina fruit were 82.89±13.12 mg/g and 14.31±9.27 mg/g respectively.Epicatechin was the basic units occurring in P.salicinafruit condensed tannins,A-type and B-type linkage were most commonlybetween the structural units of polymers.The average degree ofpolymerization （DP） of condensed tannins was 5.3,and the mean molecularweight was 1583.7.The effects of tannins from P salicina fruit on freeradical-scavenging were determined by DPPH radical scavenging activity andthe IC₅₀ value was 57.98μg/mL.（2） Hydrolysable tannins in S.cuminic fruitstone were indentified as ellagitannins that were composed of gallic acid andellagic acid,linked to a sugar moiety.Condensed tannins in S.cuminic fruitskin were identified as B-type oligomers of epiafzelechin （propelargonidin） witha degree of polymerization up to eleven.The antioxidant activity,measured bytwo vitro models:DPPH radical scavenging activity and ferricreducing/antioxidant power.Tannins extracted from S.cumini fruit showed avery good DPPH radical scavenging activity and ferric reducing/antioxidantpower.The results indicate promising the fruit of S.cumini for the utilization assignificant source of natural antioxidant.（3） The predominance of signalsrepresentative of procyanidins and prodelphinidins with 2,3-cisstereochemistry of condensed tannins was determined in the stem bark of C. album.In addition,epicatechin and epigallocateclin polymers with galloylatedprocyanidin or prodelphinidin were also observed.The average DP and theaverage molecular weight were 5.3 and 1578.25,respectively.TheMALDI-TOF MS,NMR and HPLC provide the rapid and ideal method forcharacterization of polydispersed vegetable tannins.Tannins extracted fromleaves,twigs and stem bark all showed very good DPPH radical scavengingactivity (IC₅₀ of 56.86,62.31 and 54.80μg/mL) and ferric reducing power （4.28,3.74 and 4.49 mmol AAE/g dried tannins）.更多还原