【作者】 靳利娥；

【导师】 常丽萍；

【作者基本信息】 太原理工大学 ， 化学工程与技术， 2010， 博士

【摘要】 生物体内存在的过量氧自由基与蛋白质、脂类化合物、多糖、核酸等大分子发生氧化反应造成对细胞损伤和引起组织结构的非正常变化,进而引发心血管疾病、衰老甚至癌变等问题。抗氧化性物质可淬灭自由基,阻止因过量自由基和生物大分子作用发生的一系列链式氧化过程。尽管通过化学手段合成的物质如叔丁基羟基茴香醚(BHA)、2,6-二叔丁基对甲酚(BHT)、没食子酸丙脂(PG)、叔丁基对苯二酚(TBHQ)等具有优良的抗氧化性质,且在食品及其它领域已经得到广泛应用,但近年研究发现,这类物质对人体呼吸酶活性产生一定不利影响,有的甚至还有致畸作用,一旦通过各种途径进入人体会产生潜在危害。因而寻找安全、可靠的抗氧化物质的成为近年世界各国精细化工领域研究的重点内容之一。许多物种尤其是食品类植物资源中均不同程度地含有一定量的抗氧化性物质,通过科学提取可得到比化学法所合成的抗氧化物质相对更为安全和可靠的天然抗氧化活性物质。然而,提取的天然抗氧化性物质是一种混合物,其抗氧化效果往往弱于合成物,热稳定性也较差,尤其是提取物中的超氧化物歧化酶(Superoxide Dismutase, SOD)、过氧化物酶(Peroxidase, POD)等,由于它们都是蛋白酶,对环境要求比较苛刻,从而影响抗氧化活性和稳定性以及储存的时间,使天然抗氧化物的应用受到一定限制。鉴于这些缺点,本实验除对提取抗氧化活性物质的方法进行了系统研究之外,还考察了某些效应物存在对抗氧化物酶的影响以及人体所需的微量金属元素同所提取的非酶天然提取物的抗氧化活性之间的协同作用,选择出了稳定性高、抗氧化性较强的“复合型抗氧化物”。实验选用资源比较丰富的茄科植物茄茎、枸杞、辣椒为原料,采用索氏提取、浸渍和超声波等方法提取非酶抗氧化性物质,用盐析法、有机溶剂沉淀法及超滤法分级分离等手段提取抗氧化物酶,确定出最佳提取方法、条件,用统计学原理分析实验结果是否存在差异性显著及可靠性,同时对不同提取物和加入一定效应物后的抗氧化性进行评价,考察了效应物金属离子、VC、VE和一些有机物对抗氧化提取物热稳定性和储存稳定性产生的影响,为开发安全可靠的天然复合型抗氧化性物提供科学指导和帮助。主要结论如下：(1)以过氧化值(POV)为评价指标选择了非酶提取物最佳提取条件：对于辣椒非酶提取物,以85%乙醇为溶剂,采用超声波法超声30 min得到的提取物抗油脂能力最强；对于枸杞非酶提取物,先以60%乙醇为溶剂,通过超声波法超声40 min,在pH为1-2的水溶液条件下水解3h后效果最好；对茄茎非酶提取物,用60%乙醇为溶剂,采用浸渍法提取。提取方法同样影响酶类提取物的提取条件：体积为原料二倍的水为溶剂超声波(40kHz)法低温下超声30 min得到酶的初提物,再经过硫酸铵分级分离、超滤精制后的抗氧化物酶SOD；对抗氧化物酶POD,除进行硫酸铵分级分离和超滤外,还需经过使用丙酮进行分级分离；和对照组对提取方法进行t-检验,差异性显著。选择原料实验表明：以辣椒为原料提取POD和以枸杞为原料提取SOD。(2)不同原料得到的提取物抗氧化性能在不同评价指标中因作用机理不同表现出不同的抗氧化性。茄茎非酶提取物对DPPH-有很强的清除能力,其抗油脂氧化性比VC、柠檬酸和VE强,但比BHT的差；辣椒非酶提取物在抗p-胡萝卜素氧化能力、清除超氧阴离子自由基能力和抗油脂氧化能力方面均比BHT强,但在对DPPH-清除能力方面表现较弱；枸杞非酶提取物在抗p-胡萝卜素氧化能力方面和BHT相当,在对DPPH-清除能力方面表现具有较强的作用。辣椒酶提取物的POD在温度为60℃,pH为5时活力最大,平均活力为16.77 u/g,通过Dalziel和Alberty方程对POD催化的反应进行了数据拟合,证明POD酶催化反应为有序反应；枸杞提取物的SOD在45℃、pH为6.5时活力最大,平均活力为171.8 u/mL。(3)采用气相色谱-质谱联用技术(GC-MS)对非酶提取物活性成分测定结果表明：茄茎提取物主要成份有棕榈酸、苯乙胺、苯甲酸、邻苯二甲酸二丁酯等芳香族和有机酸类化合物以及少量酚类化合物；辣椒提取物主要含辣椒素、2,4-癸二烯醛,9,12-十八碳二烯酸,辣椒碱,苯乙酸,4-羟基-3-甲基酚等酚类、脂肪酸等化合物；枸杞非酶提取物主要有氧化萜类、脂肪酸脂类、醛酮类、芳香族化合物。非酶提取物中总酚含量与其产率之间的没有显示出相关性(r2=0.1813),与POV值之间线性相关性也较差(r2=0.492)。但和DPPH-抑制率以及p-胡萝卜素脱色率之间存在较强的线性相关,其相关系数分别为r2=0.9927和r2=0.9855,t-检验也证实结果间差异性显著。(4)提取物与效应物协同实验表明：添加不同效应物,表现出的效果差别较大。非酶提取物和Vc按1：1混合,其抗氧化性优于合成抗氧化剂BHT；微量金属离子的存在对非酶提取物抗油脂氧化能力、清除O2-·和DPPH·能力、抗p-胡萝卜素氧化率等均有一定程度的影响；其中以Se(Ⅳ)在辣椒和枸杞非酶提取物中的存在均能显著提高提取物的抗氧化能力,表现出明显增效作用,说明二者存在一定的协同关系；当Fe2+和Mn2+浓度分别为800 mg/L和500 mg/L时,枸杞非酶提取物对O2-·自由基清除率可分别提高55%和40%,辣椒非酶提取物对O2-.自由基清除率可分别提高81%和79%。相同效应物对辣椒POD和枸杞SOD活力影响不同。KCl、NaCl、CaCl2、ZnSO4、MgSO4、乙酸锌、葡萄糖酸锌、葡萄糖酸锰、乙二醇、丙三醇、D-甘露醇和D-山梨醇对POD为激活剂,CuCl2、柠檬酸对POD为抑制剂,对活力影响符合“S”型曲线规律。浓度为2.0 m mol/L、混合比例为1：1的乙酸锌和D-山梨醇组成的二元效应物可提高80%的POD活力。乙二醇、丙三醇和D-山梨醇对SOD为激活剂,KCl、NaCl、CaCl2、ZnSO4、MgCl2、乙酸锌、葡萄糖酸锌和葡萄糖酸锰为抑制剂,对活力影响符合“双曲线”型规律,利用双倒数法确定出葡萄糖酸锌、乙酸锌、Ca2+、Mg2+属于竞争性抑制剂,而K+、Na+、Mn2+、Cu2+、ZnSO4属于反竞争抑制剂。(5)建立了辣椒和枸杞非酶提取物失活速率方程,得到非酶提取物在效应物亚硒酸钠存在情况下失活反应活化能变低,稳定性减弱。对辣椒非酶提取物,其失活速率k=1.42×102.e(?),r2=0.99；加入亚硒酸钠效应物后的反应速率k=0.167.e(?),r2=0.958,活化能降低了53.8%。对枸杞非酶提取物,失活速率符合方程k=1.519.e(?),r2=0.908；加入亚硒酸钠效应物后,反应速率k=1.669×10-2·e(?),r2=0.981,活化能也变小。对于辣椒POD,失活速率符合k=1.36×109e(?),r2=0.960,加入效应物后,失活速率符合k=3.406×108e(?),r2=0.967,加入效应物后POD失活反应的活化能增大；对于枸杞SOD,k=3.612×103e(?),r2=0.972,加入效应物后,k=2.355×104e(?),r2=0.904,活化能亦增加。计算表明,在室温4℃下辣椒POD的抗氧化活性损失是25℃时活性的十分之一,枸杞SOD在4℃时的活性损失是25℃时的四分之一,加入效应物后可使半衰期延长一倍。(6)研究了POD和SOD的微电泳行为和POD电化学稳定性的规律。结果表明,在pH为2-9的范围,POD和SOD均带负电荷；未加入效应物前颗粒之间相互吸引作用较强,团聚作用明显,颗粒尺寸分布宽,平均粒径较大；加入效应物后颗粒尺寸分布变窄,平均粒径较小。POD的等电点(pI)约等于5.19,SOD的pI值约等于5.1；加入效应物后的POD和SOD的pI位移量大,保持较高的稳定性；用纤维素包埋法将效应物-辣椒过氧化物酶-纤维素／多壁碳/Pt的复合物修饰在Pt片电极表面,制备成的修饰电极稳定性良好,得到的循环伏安曲线峰形最好,峰电流大。更多还原

【Abstract】 Superfluous free radicals called refuse in organism can have much effect on giant molecules such as protein, lipoid, polysaccharide, nucleic acid. It dam-ages the activity and the structure of normal cell tissue. This will cause many diseases like angiocardiopathy, aging, transformation of normal cells into can-cerous tumor cells, etc. Therefore, getting rid of these superfluous free radicals plays an important role on maintaining body health. Antioxidant may destroy these superfluous free radicals and break the process of chain reaction. The an-tioxidant substances synthesized by chemical method like butylated hy-droxyanisole (BHA), propyl gallate (PG), tertiary butylhydroquinone (TBHQ), etc., which performed excellent activities, have been widely used to food in-dustry, cosmetics and other fields. However, recent-studies showed that synthe-sized antioxidants might have an unfavorable influence on respiratory ferment. Some even cause potential teratogenesis. This is to say, once they get into the body our health will be threatened. Obviously, searching for safe and reliable antioxidants becomes one of the emphatically studying contents for the fine chemicals in the world in recent years. Many plants, especially for the solana-ceae plants resources, contain some antioxidants in various degrees. They can be extracted by scientific way and have safer and more trustful characteristics. It is noticed that extracted antioxidant from the plants are a kind of mixture. Its antioxidant activities are usually weak relative to synthesized antioxidant. And its thermal stability isn’t good, especially for the extracts like superoxide dis-mutase (SOD), peroxydase (POD), which is apt to degenerate. These problems seriously hinder the wide application of natural antioxidant. In view of this point, the experiment observed the influences on natural antioxidant activities in the existence of necessary metal elements in the body after systematically studying extraction ways on yields so that obtain dual function antioxidant, which both has antioxidant activities and increases the trace elements, also.The experiments used solanaceae plants such as stalk of S.melongena, ly-cium, and chilli as materials, which have an abundant resource. By experiment, we investigated three extracting ways like extraction, impregnation and ultra-sonic wave on extraction yield of non-enzyme antioxidant and salting out method, precipitation with organic solvents, ultrafiltration fractionation on ex-traction yield of enzyme antioxidant. The optimal extracting method and condi-tion are given. At the same time, the antioxidant activities of enzyme extracts after adding some effectors such as metal ions, Vc, VE and some organic sub-stance were determined and analyzed. The optimal ratios of extracts to effects were chosen. In addition, the thermal stability and storage time for these ex-tracts were also observed. The experimental results were analyzed by general theory of statistics, which showed whether the experimental result had signifi- cant difference and its reliability. The main contents are as following:(1) Extracting methods and types of solvent both affect the effectiveness of anti-oxidant. For chilli, optimal extracting condition is that 85% ethanol is used as extracting solvent, extracting time is 30min by ultrasonic wave. For lyceum, 60% ethanol was selected as solvent, ultrasonic wave irradiating time is 40 min, then, hydrolysis time for the extracts is 3h; For stalk of S.melongena,60% etha-nol is used as solvent and impregnating extraction is selected.The extracting methods also have an influence on activity of superoxide dismutase (SOD) and peroxidase (POD). The activity of SOD obtained through ultrasonic wave extraction, salt out of ammonium sulphate and hyperfiltration is t he highest; the activity of POD obtained through extraction of water, salt out of ammonium sulphate, treatment of acetone and hyperfiltration is the highest. T-test showed that there existed significant difference for these methods. The activity of SOD and POD produced by extracting different materials is not the same. The activity of SOD from chilli is lower than that of lyciun Activity of POD from chilli is stronger than that of lycium.(2) For the same extract, anti-oxidizing property is different due to using different evaluating methods. The non-enzyme extracts from stalk of S.melongena has strong clearing ability for DPPH-, but the ability of anti-oxidization of oil is poorer than BHT. The non-enzyme extracts of chilli has strong abilities in respect of anti-oxidation forβ-renieratene, clearing ability of superoxide anion free radical and anti-oxidation of oil than BHT, but has weak ability for clearing DPPH-. The non-enzyme extracts of lycium has not only strong ability for clearance of DPPH-, but also anti-oxidation for P-renieratene is equivalent to BHT. For SOD the optimal temperature is at 55℃and the optimal pH is 6.5; for POD the temperature is 45℃and pH is 6.0.(3) It was shown by gas chromatography-mass spectrum (GC-MS) that the main composition of the non-enzyme extracts in stalk of S. Melongena are aro-matic series and organic acid compounds such as hexadecanoic acid, phenethylamine, phenylformic acid, dibutyl phthalate, and a small amount of phenolic compounds; for the extracts of chilli, the main compositions are phe-nols and fatty acid compounds like capsaicin,2,4-decadienal,9,12-octadecadi-enoic acid, capsicine, phenylacetic acid,4-hydroxy-3-methylphenol; for the ex-tracts of lycium, the main compositions are such compounds as Oxidation of terpenes, fatty acid ester, aldoketones, aromatic series. There wasn’t an obvious dependence between the content of the total phenol in the non-enzyme extracts and yields. However, the suppression ratio of DPPH·and decolorization ratio ofβ-renieratene for non-enzyme extracts seriously depend on the content of the total phenol whose the related coefficient r2 are 0.9927 and 0.9855, respectively. This conclusion is also proved by T-test.(4) The synthetic experiments of extracts and effectors showed that differ-ent types of effectors displayed a great difference. For the non-enzyme extract, trace metal ions can improve the ability of antioxidant, eliminating peroxy radi-cal and DPPH, and oxidation resistance of p-renieratene. The antioxidant of the mixture composed of non-enzyme extracts and vitamin C, which account for 50 wt% in the mixture, is more excellent than synthetics BHT. The antioxidant of non-enzyme extracts for chilli and lycium is greatly enhanced in the existence of selenium ion, which illustrated the existence of synthetic effect. When the concentration of Fe2+ of 800mg/Land Mn2+ of 500mg/L are used and other con-ditions are the same, clearance ability for superoxide radical increases by 55% and 40% for the non-enzyme lycium extracts, and 81% and 79% for the non-enzyme chilli extracts, respectively.Influence of the effectors on activity of POD and SOD isn’t the same even if the type of effectors is the same. Such effectors as potassium chloride, sodium chloride, calcium chloride, barium chloride, zinc sulfate, magnesium chloride, zinc acetate, zinc gluconate, manganese gluconate, glycol, propanetriol, mannite and sorbierite are the activating agents for the activity of POD. Such compounds as copper chloride, citric acid are the inhibitors for the activity of POD whose affected tendency conforms to the curve S shape. Binary effectors composed of zinc acetate and sorbierite,which accounts for 50% and its concentration of 2.0 mmol/L is used, increase by 80% activity of POD. Such compounds as glycol, propanetriol and sorbierite are activating agents for the activity of SOD. Such compounds as potassium chloride, sodium chloride, calcium chloride, barium chloride, zinc sulfate, magnesium chloride, zinc acetate, zinc gluconate, man-ganese gluconate are the inhibitors for the activity of SOD. Furthermore, through binary counting backward technique of Michaelis-Menten Equation confirmed that influence of the compounds like zinc gluconate, zinc acetate, calcium chloride, barium chloride, magnesium chloride on SOD belongs to the competing inhibitors and compounds such as potassium chloride, sodium chlo-ride, manganese gluconate, copper chloride and zinc sulfate belongs to anti-competitive inhibitors.(5) The reactive rate equation occurred in the process of oxidizing inactiva-tion for the non-enzyme extracts of chilli and lycium were given out, from which the activation energy of reaction is reduced in the existence of effectors sodium selenite. This can strengthen the ability of getting rid of free radicals of non-enzyme extracts. For non-enzyme extracts of chilli, its rate equation of in-activation is k=1.42×102·e (?), corresponding related coefficient r2 equals 0.99. After effectors sodium selenite was added, rate equation of inactivation is k=0.167·e (?) and related coefficient r2 is 0.958. The activation energy de-creases by 53.8%. For non-enzyme extracts of lycium, inactivation rate kis 1.519·e RT and related coefficient r2 equals 0.908. When effectors was used the inactivation rate k becomes 1.669×10-2·e RT and related coefficient r2 equals 0.981. The activation energy also decreases.For the POD extracted from chilli, its inactivation rate k conforms to 1.36×109·e (?) and related coefficient r2 is 0.96. Once effectors sodium selenite was added into the extracts, inactivation rate k becomes 3.406 x108·e (?) and related coefficient r2 is 0.972. The inactivation energy increases; For SOD, inactivation rate k conforms to 3.612 x103·e (?) and related coefficient r2 is 0.972. After the effectors sodium selenite was added, inactivation rate k is ex-pressed as 2.355×104·e (?). And the related coefficient r2 is 0.904. The activa-tion energy also increases. The activity of POD and SOD was seriously influ-enced by the temperature and easily becomes inactive. The calculation showed that the activity of antioxidant for the chilli extracts at the temperature of 25℃is one tenth of the activity at 4℃because of the loss of activity. For SOD of ly-cium extracts, its activity is one fourth of the activity at the temperature of 4℃due to the loss.(6) The electrical behaviors of POD and SOD were also studied. The re-sults showed that POD and SOD exist with a negative charge in the range of 2 to 9 for pH. The force produced among the particles behaviors strong, and the ag-gregation becomes obvious. The distribution range of the particle size is wider and average diameter is larger; When the effectors were added the distribution range of the particle size is narrow and its average diameter becomes small. The amount of displacement is larger which is beneficial to keeping the extracts sta-ble. And cyclic voltammogram showed that the stability of the electrical polar manufactured by POD-MC/MWCNTs/Pt peak shape is better and peak electrical current is big.更多还原