【作者】 顾海峰；

【导师】 李春美；

【作者基本信息】 华中农业大学 ， 食品科学， 2007， 硕士

【摘要】 本文采用无水甲醇为提取溶剂从柿子果肉中提取单宁，通过考察提取剂种类、提取温度、时间、提取次数及料液比等因素来确定了柿子单宁的最佳提取工艺；采用大孔树脂101结合超滤分离实现了柿子单宁的分离纯化及分级制备，得到了高分子量单宁（HMWT）和小分子量单宁（SMWT）两个级分。采用高效液相-电喷雾质谱联用（HPLC-ESI-MS）对小分子结构进行了初步鉴定；采用凝胶渗透色谱（GPC）、傅立叶红外光谱（FT-IR）以及硫解和酸解结合HPLC-ESI-MS等方法分析了HMWT分子量分布以及单体结构；并对HMWT与SMWT在不同体系中的的抗氧化活性及HMWT与牛血清白蛋白（BSA）的相互作用进行了系统研究；同时重点研究了HMWT在体外对江浙蝮蛇毒（AhPVP）、尖吻蝮蛇毒（AaVP）、眼镜蛇毒（NnaVP）中蛋白酶活的抑制能力及体内对AhPVP、AaVP、NnaVP染毒小鼠的解毒效果；并结合SDS-PAGE凝胶电泳、红外光谱（FT-IR）、圆二色谱（CD）初步研究了HMWT与蛇毒蛋白的相互作用机理。主要研究结果如下：1．柿子单宁的最佳提取工艺为：新鲜果肉去皮打浆后，以含1％HCL的无水甲醇作为提取介质，料液比1∶12，90℃加热回流提取，提取3次，每次40min。2．提取液经大孔树脂101除糖去杂，聚砜超滤膜分级得到红色的HMWT粉末以及淡黄色SMWT粉末，HMWT缩合单宁（CT）含量93.4％，SMWT总酚（TP）含量为87.4％。两者具有典型的酚类物质显色特征，易溶于水，乙醇，甲醇等溶剂，其中HMWT在水中的最大溶解度为100mg／mL。3．经HPLC-ESI-MS鉴定SMWT主要为没食子酸及分子量分别为461，461，603的三个未知化合物。HMWT分子量分布在1.16×10⁴u-1.54×10⁴u之间，重均分子量1.39×10⁴u，数均分子量1.28×10⁴u，FT-IR表明HMWT具有原花青素的基本骨架结构，硫解和酸解的HPLC-ESI-MS表明其主要组成单体为：表没食子儿茶素没食子酸酯（EGCG），表儿茶素没食子酸酯（ECG），（表）没食子儿茶素（E）GC及一种未知结构的单体。4．HMWT在2-脱氧-D-核糖体系，甲基紫体系，水杨酸体系中对羟基自由基的最大抑制率分别为90.47％、96.46％和87.77％；对邻苯三酚自氧化以及亚油酸脂质过氧化的最大抑制能力分别为56.57％和69.63％。在所有的体系中HMWT的抗氧化能力与加入量呈明显的剂量效应关系，且HMWT的抗氧化能力要优于相同浓度下的葡萄籽原花青素，大大强于SMWT，是柿子单宁抗氧化作用的主导成分。5．HMWT与明胶和BSA均具有较强的结合能力，明胶、BSA与HMWT质量比达到1.6∶1时单宁沉淀率达到最大。HMWT与BSA结合的适宜条件为：温度40℃，反应时间30min，体系pH5.0，0.5mol／L的NaH₂PO₄为溶解介质。荧光光谱和紫外吸收光谱法分析表明，HMWT可以有规律地使BSA内源荧光猝灭，其猝灭机理可认为是HMWT与BSA形成复合物的静态猝灭、HMWT与BSA的作用力为疏水作用力和静电作用力，同时由F（?）rster非辐射能量转移理论计算得出HMWT与BSA结合位置的距离为1.63nm，结合位点0.99。6．HMWT溶液在光照、温度大于60℃、pH大于7条件下稳定性较差；具有较好的抗氧化性和抗还原性；食品添加剂中β-环状糊精有利于增加HMWT的热稳定性、淀粉在溶液中含量超过O.5％时易与HMWT发生复合作用，柠檬酸三钠对稳定性有较大的影响，柠檬酸、EDTA则影响较小；金属离子中Fe³⁺，AL³⁺，Zn²⁺对HMWT有着较强的结合作用，Na⁺、K⁺、Mg²⁺、Ca²⁺、Cu²⁺则影响较小。7．当蛇毒蛋白与HMWT质量比为1∶1时，HMWT对AhPVP、AaVP、NnaVP中蛋白质水解酶、磷脂酶A₂、L-氨基酸氧化酶、精氨酸酯酶、乙酰胆碱酯酶、类凝血酶的酶活均基本完全抑制，且呈明显的剂量效应关系。8．HMWT对昆明小鼠腹腔注射的LD₅₀值为301.84mg／kg，具有良好的安全性。当蛇毒蛋白与HMWT质量比为1∶1时，HMWT对AhPVP，AaVP，NnaVP引起的肌肉毒性有极显著（p＜0.01）的抑制作用，肌酸激酶（CK）的酶活抑制率分别为63.30％，55.06％，61.82％，肌肉组织病理学观察也显示出明显的剂量效应关系，高剂量组小鼠肌纤维损伤明显好转。当蛇毒蛋白与HMWT质量比为1∶1时，HMWT能够极显著（p＜0.01）降低AhPVP，AaVP引起的皮下出血，出血抑制率分别为98.6％，96.9％，HMWT对出血的抑制呈明显的剂量效应关系。9．5LD₅₀剂量的AhPVP、AaVP、NnaVP注射入昆明小鼠腹腔后，口服灌胃HMWT后可使AhPVP、AaVP和NnaVp所致的染毒死亡小鼠平均存活时间分别延长4-6倍、3-5倍和2-4倍，高剂量的HMWT可使AhPVP、AaVP和NnaVp所致的染毒小鼠存活率分别提高到75％、50％和25％。腹腔注射HMWT后可使AhPVP、AaVP和NnaVp所致的染毒死亡小鼠平均存活时间分别延长15-30倍、15-20倍和5-15倍，高剂量组对各种蛇毒的解毒作用强，小鼠存活率达100％。静脉注射HMWT对小鼠的急救效果最佳，HMWT中剂量组即可使各组小鼠存活率达100％，低剂量组也能大大延长存活时间并且存活率分别达到了50％、75％、50％。三种解救方式对蛇毒引起的毒害均有急救效果，口服灌胃虽然存活率不高但能有效的延长存活时间，而腹腔注射和静脉注射则能有效提高存活率。口服预防给药HMWT可使AhPVP、AaVP和NnaVp所致的染毒死亡小鼠平均存活时间分别延长3-5倍、3-5倍和1-3倍，说明服用HMWT对蛇毒引起的毒害有一定的预防作用。10．SDS-PAGE结果表明HMWT与蛇毒蛋白结合形成沉淀是导致酶失活的重要原因，虽然HMWT对蛇毒中蛋白质组分的结合表现出一定的非特异性，但对不同组分结合能力和选择性明显不同，且为非可逆结合。AhPVP经Sephadex G-75两次纯化及DEAE Sephadex A-50离子交换层析得到分子量14000的电泳纯磷脂酶A₂。红外及圆二色谱结合表明，HMWT中的酚羟基与PLA₂中的氨基的相互作用在结合过程中起着重要的作用，且HMWT对PLA₂的a-螺旋结构有强烈的破坏作用。更多还原

【Abstract】 Persimmon tannin was extracted from persimmon pulp by anhydrous methanol, the effects of pre-treating, extraction temperature, time, extraction times, solid/solvent ratio and concentration of HCl on the yield of tannin were studied and the optimal extracting conditions was obtained. Macroporous adhesive resin 101 and Ultra filtration were employed to separate and fractionate the crude extract and two fraction: Small Molecular Weight Tannin （SMWT） and High Molecular Weight Tannin （HMWT） were obtained.. The structure of SMWT was identified by HPLC-ESI-MS. The molecules weight distribution of HMWT was determined by GPC and its thiolysis degradation and acid hydrolysis products were identified by HPLC-ESI-MS to confirm its monomer composition. Antioxidant activity of HMWT and SMWT in different systems, and aggregation behaviors of Bovine Serum Albumin （BSA） with HMWT, as well as their interaction manner was studied systemically. Inhibiting effect of HMWT on enzyme activities from Agkistrodon halys Pallas Venom Protein （AhPVP）, Agkistrodon acutus Venom Protein （AaVP） and Naja naja atra （NnaVP） Venom Protein and detoxic effect of HMWT were investigated in vitro and in vivo, respectively. Interaction mechanism of Venom Protein and HMWT were studied by SDS-PAGE, FT-IR, and CD. The main results were shown as follows:1.The optimum extraction conditions of persimmon tannin were anhydrous methanol containing 1% concentrated HCl as extraction media with a solid/solvent ratio of 1:12 （w/v）, extraction at 90℃for 40min and extracting for three times.2. Extraction solution was adsorbed on Macroporous adhesive resin 101 to remove carbohydrate and other impurity and polysulfone ultrafilter membrane partition to yield two fractions: yellowy SMWT powder and red HMWT powder. The condensed tannin （CT） content in HMWT was 93.4% and total polyphenol （TP） content in SMWT was 87.4%. Both of them possess typical chromogenic reaction of polyphenol and were found to be eutectic in water, ethanol, methanol and the highest solublity of HMWT in water reached 100mg/mL.3. HPLC-ESI-MS. analysis showed that SMWT was composed of gallic acid and other three unknown compounds with molecular weight of 461, 461 and 603, respectively. The molecules weight distribution of HMWT was determined to be 1.16x10⁴-1.54x10⁴u with the （?）=1.28x10⁴u and （?）=1.39x10⁴u by GPC. FT-IR revealed that HMWT had typically characteristic absorptions of basic proanthocyanid structural. Its thiolysis degradation and acid hydrolysis products were identified to be: （epi） gallocatechin, epigallocatechin-3-O-gallate, epicatechin-3-O-gallate, and one unknown monomer by HPLC-ESI-MS.4. The maximum scavenging rate of HMWT on hydroxyl radical in 2-deoxy-D-Ribose system, methyl violet system and salicylic acid system were 90.47%, 96.46% and 87.77%, respectively; the maximum inhibiting effect of HMWT on pyrogallol autoxidation and linoleic acid lipid preoxidation were 56.57% and 69.63%, respectively. HMWT exhibited excellent antioxidant activity in all tested systems in a dose-dependent manner and the antioxidant activity of HMWT was significantly stronger than those of SMWT and grape seeds proanthocyanidin. These results suggested that HMWT is the main antioxidant components in persimmon pulp.5. HMWT was found to have strong aggregation ability with glutin and BSA, the precipitation rate reached maximum when the rate of glutin and BSA to HMWT reached 1.6:1（w/w）. The feasible aggregation conditions of HMWT with BSA were 0.5mol/L NaH₂PO₄ as dissolution media, temperature at 40℃for 30min and the system pH was 5.0. The interaction between HMWT and BSA were studied using fluorescence spectra and ultraviolet-visible light absorption spectra. The results showed that the fluorescence of BSA was quenched regularly by HMWT and the type of quenching was the static quench. The binding constants and thermodynamic parameters of HMWT with BSA were obtained at different temperatures. The hydrophobic interaction force and electrostatic force played a main role in the binding of HMWT with BSA. The distances （1.63nm） of binding site （n=0.99） between HMWT and BSA were obtained according to the theory of Forster’s non-radiative energy transfer. 6. HMWT was unstable when exposed to light、above 60℃or in alkaline conditions; HMWT had relatively good antioxidant and anti-reduction property;β-cyclodextrin could improve the heat stability of HMWT, starch could form complex with HMWT when its concentration reach 0.5% in solution, Trisodium citrate had effects on the stability of HMWT; Other food additives such as citric acid and EDTA had little effect on its stabilityt; HMWT was unstable on Fe³⁺, Al³⁺ and Zn²⁺ while Na⁺、K⁺、Mg²⁺、Ca²⁺、Cu²⁺ had no obvious effect on its stability.7. The activities of proteolytic enzyme, phospholipase A₂, L-amino acid oxidase, arginine esterase, acetylcholinesterase, and thrombin like enzyme from AhPVP, AaVP and NnaVP were inhibited completely when the rate of snake venom to HMWT reached 1:1 （w/w）, and the inhibiting effect was in a dose-dependent manner.8. Acute toxicity tests on HMWT showed that it’s LD₅₀, i. p mice 301.84mg/kg. Myotoxicity from AhPVP, AaVP and NnaVP were inhibited statistically significant（p＜0.01） when the rate of snake venom to HMWT reached 1:1 （w/w）, the inhibiting effect of HMWT on creatine kinase（CK） activities were 63.30%, 55.06% and 61.82%, respectively; The muscle tissue pathological changes were improved and repaired obviously at the dose of 1:1 （w/w）. Hemorrhage lesion from AhPVP and AaVP were inhibited statistically significant（p＜0.01） when the rate of snake venom to HMWT reached 1:1 （w/w）, the inhibiting effect of HMWT on hemorrhagic spots area were 98.6% and 96.9%, respectively and both in dose-dependent manner.9. AhPVP、AaVP、NnaVP were separately injected into mice paunch in the dose of 5LD₅₀ and their average live time were 24.50min, 35.50min and 19.50min, respectively. Therapeutic administration by i. g of HMWT could postpone the average live time of dead mice caused by AhPVP、AaVP、NnaVP by 4-6 times, 3-5 times and 2-4 times, respectively And administration HMWT with a dose of 75LD₅₀ could increase the survival rate to 75%, 50% and 25%, respectively. Therapeutic administration by i. p of HMWT could postpone the average live time of dead mice caused by AhPVP、AaVP、NnaVP by 15-30 times, 15-20 times and 5-15 times, respectively, and administration HMWT with a dose of 15LD₅₀ HMWT the survival rate in every group reached 100%. When therapeutic administration by i. v of HMWT, the survival rate of mice caused by AhPVP、AaVP、NnaVP reached 50%、75%、50%, respectively, and administration HMWT with a dose of 5LD₅₀ HMWT the survival rate in every group reached 100%. Three first aid method by administration of HMWT were effective to increase survival rate or prolong the live time of the mice executed by AhPVP、AaVP and NnaVP.Preventive administration （by i. g） of HMWT could postpone the average live time of dead rats caused by AhPVP、AaVP、NnaVP by 3-5 times, 3-5 times and 1-3 times, respectively.9. The SDS-PAGE electrophoretograms demonstrated that the precipitation of the venom-HMWT mixture happened in a dose-dependent manner which corresponded to the inhibition effect of enzyme activities. Their interaction was a non-specifically, irreversible manner, but with different selectivity for different protein. The PLA₂ with molecular weight 14000u was isolated from AhPVP by successive Sephadex G-75 and DEAE Sephadex A-50 chromatographies. FT-IR and CD analysis demonstrated that the interaction between hydroxyl from HMWT and amidocyanogen from PLA₂ play an important role in the combine process, and HMWT had strong configurational changes to a-helix of PLA₂.更多还原