灵芝液体发酵清除自由基活性产物发酵条件的优化

以红灵芝(Ganoderma.lucidum)为实验菌株,对其液体发酵活性产物清除自由基的发酵条件进行了研究。实验考察了培养基成分对灵芝液体发酵所产生的有效产物清除自由基能力的影响。结果表明,葡萄糖、酵母粉分别为灵芝发酵活性产物清除自由基效果较合适的碳源、氮源,最佳质量浓度分别为40 g.L-1和3 g.L-1,清除自由基的能力分别为55.7%、40.8%和66.5%、50.6%;铁离子对灵芝发酵产物清除超氧阴离子具有明显的效果,适宜质量分数为70×10-6;清除超氧阴离子的能力为54.6%,而硒离子对灵芝发酵产物抑制羟自由基有明显的促进作用,最适的质量分数为90×10-6清除羟自由基的能力为67.7%。油酸能促进灵芝发酵产物的清除超氧阴离子和羟自由基的能力,适宜的质量分数为0.1%,清除两种自由基的能力分别为:73.7%,53%。     

茶儿茶素氧化机理

在制茶发酵过程中,儿茶素可发生苯骈环化反应而形成茶黄素,茶黄素还可转化成茶萘酚醒、脱氢茶黄素和高聚合物;同时,儿茶素可发生二聚合反应产生原花青素类以及双黄烷醇类等物质;儿茶素还可能与茶没食子素、杨梅甙、阿福豆素、维生素C、茶氨酸发生聚合反应,形成加合物。体外自由基清除实验表明,儿茶素具有优良的抗氧化特性。儿茶素在清除自由基时,自身氧化产生双黄烷醇,也可发生A环的断裂而产生羧酸类物质。     

苦皮藤中酚性成分抗羟自由基活性研究

从苦皮藤中分离出四个酚性成分 :( )儿茶素 ( 1) ,(— ) -表儿茶素 ( 2 ) ,3,4 ,5-三甲氧基 -苯 - 1- O- β- D-葡萄糖甙 ( 3)和 3,7,4 -三羟基 - 3 -甲氧基黄烷 - 5- O- β- D-葡萄糖甙 ( 4 )。其中 ( 4 )为新化合物。用电子自旋共振技术 ( ESR)研究了它们对羟自由基即(· OH)的清除作用。     

黄伞胞外多糖抗自由基作用的研究

目的:以乙醇沉淀的黄伞发酵浓缩液得到的胞外多糖为研究对象,检验其对自由基的清除作用。方法:采用电子顺磁共振(EPR)波谱仪检测黄伞胞外多糖清除羟自由基OH.和超氧阴离子自由基O2-.的作用。结果:在样品浓度均为100mg/mL的条件下,利用Fenton反应体系,黄伞胞外多糖对羟自由基OH.的清除率仅为38.5%;而利用次黄嘌呤-黄嘌呤氧化酶体系,黄伞胞外多糖对超氧阴离子自由基O2-.的清除率可达到80.6%。结论:黄伞胞外多糖在体外对自由基有一定的清除作用且其对超氧阴离子自由基O2-.的清除作用明显优于对羟自由基OH.的清除作用。     

3种百合鳞茎中多酚类物质的抗氧化活性分析

以野生百合渥丹、山丹和传统食用的兰州百合为研究对象,对其鳞茎中多酚类物质、11种单体酚的含量及抗氧化活性(ABTS自由基、超氧阴离子、羟自由基的清除能力,铜离子还原能力以及抑制脂质过氧化活性)进行了分析。结果表明:两种野生百合鳞茎中的多酚类物质含量及抗氧化活性均显著高于兰州百合。3种百合鳞茎中单体酚的种类也有所不同,但均含有没食子酸、矢车菊素-3-芸香糖苷、儿茶素、表儿茶素、杨梅酮、芦丁、对香豆酸、山奈酚。相关性分析显示,除对羟自由基的清除力外,各酚类物质总量与不同抗氧化指标之间呈显著正相关关系。试验结果认为,野生百合鳞茎可作为天然抗氧化资源应用于食品和医药业,具有一定的开发应用前景。     

瓦宁木层孔菌中多酚和黄酮类成分分离及清除自由基活性的研究

采用硅胶和反相C18柱层析方法,首次从瓦宁木层孔菌中分离得到了5个化合物,运用NMR波谱法分析和鉴定为樱花亭、7-甲氧基二氢莰非素、二氢莰非素、4-(3,4-二羟苯基)-3-丁烯-2-酮、hispolon。并通过建立体外二苯基苦味酰基苯肼自由基（·DPPH）、超氧阴离子自由基（·O2?）以及羟自由基（·OH）发生体系,研究了5个化合物对·DPPH、·OH和·O2?的清除作用。结果表明当浓度达到100μg/mL时,化合物4-(3,4-二羟苯基)-3-丁烯-2-酮和hispolon对·DPPH清除率分别为92%和93%,对·OH的清除率分别为90%和95%,而对·O2?的清除率分别为70%和77%,略低于清除·DPPH和·OH的能力;二氢莰非素对·O2?自由基的清除率为39%,强于清除·OH和·DPPH的能力;而樱花亭和7-甲氧基二氢莰非素对3种自由基的清除率均低于30%。2个多酚类化合物清除自由基的能力均强于3个黄酮类化合物。5个化合物清除自由基能力均表现出一定的浓度依赖性。     

中药地榆提取物对自由基的清除作用

本文采用三种分光光度法,测定了中药地榆提取物不同极性溶剂萃取所得各部分对羟自由基和超氧阴离子自由基的清除作用,以BHT为参照.结果表明:中药地榆提取物各萃取部分除石油醚层外,其它各萃取部分对·OH自由基都有清除作用,且随着提取物浓度的增加,清除率逐渐增高;对O2-自由基没有明显的清除作用.其中乙酸乙酯萃取部分对·OH自由基的清除作用最强.     

微生物源性抗氧化剂体外抗氧化能力的初步研究

目的:研究微生物源性抗氧化剂的体外抗氧化能力.方法:在体外分别测定微生物源性抗氧化剂、α-生育酚(Vε)、抗坏血酸(VC)、L-硫辛酸、表没食子酸儿茶素的还原能力,羟自由基、超氧阴离子自由基和DPPH自由基清除能力及抗脂质过氧化能力,比较微生物源性抗氧化剂与其他抗氧化剂抗氧化能力.结果:微生物源性抗氧化剂有较强的抗氧化能力,体外清除羟自由基、超氧阴离子自由基、DPPH自由基能力的半数有效量(EC50)分别为184.5μg、48.7μg、66.1 μg.与常见抗氧化剂相比,微生物源性抗氧化剂对氧自由基及氮自由基都有较好的清除自由基作用.结论:微生物源性抗氧化剂体外抗氧化作用明显,有进一步开发的价值.     

钩藤水提物Batch-2对6-羟多巴胺诱导的SH-SY5Y细胞损伤的神经保护作用

以前的药理学研究表明,钩藤水溶性提取物C-MED-100^TM不仅具有抗氧化活性,而且还具有很好的DNA修复和免疫功能．Batch-2是一种新的水溶性钩藤提取物,而它的自由基清除能力及神经保护作用还未见报道．首先检测了batch-2对六羟多巴胺诱导的SH-SY5Y细胞损伤的神经保护作用机制,然后利用红外光谱、HPLC和分光光度技术对batch-2的组成成分进行了分析．结果表明,batch-2具有清除各种自由基的能力,尤其是对羟自由基的清除(25 mg/L的batch-2对羟自由基的清除率为60%),batch-2可剂量依赖性地抑制6-羟多巴胺诱导的细胞凋亡、脂质过氧化水平、线粒体膜电位的降低和细胞内活性氧和一氧化氮的增加．同时,batch-2抑制了由6-羟多巴胺诱导的SH-SY5Y细胞内iNOS和NF-κB蛋白的上调．结果表明,batch-2对六羟多巴胺诱导的SH-SY5Y细胞损伤的神经保护作用是通过清除活性氧和一氧化氮、抑制iNOS和NF-κB表达实现的．成分分析表明,batch-2中的多酚和奎宁酸含量分别为6.43%和0.095 8%．上述结果显示,batch-2的抗氧化机制部分类似于EGCG．对于帕金森病的预防,batch-2是一个潜在具有很好的神经保护作用的天然抗氧化剂．     

儿茶素单体EGCG酶法修饰研究及其产物抗氧化活性评价

利用Novozym 435脂肪酶在非水介质中催化儿茶素单体EGCG(表没食子儿茶素没食子酸酯)的酶促酰化反应,以增加EGCG的脂溶性。探讨了溶剂种类、水活度、加酶量、反应时间、反应温度、酰基供体等条件对酰化反应的影响。借助液质联用仪及红外光谱仪对合成产物进行鉴定,表明在叔戊醇体系中,脂肪酶可催化EGCG与丁酸乙烯酯的反应,酰化后EGCG主体结构不变,其分子中引入了四碳链的烷基。对修饰后EGCG抗氧化活性的评价表明:在相同的添加量下,酶修饰EGCG活性略低于未改性EGCG,但是清除DPPH·、O-·2自由基能力总体高于TBHQ、维生素C,清除·OH自由基能力低于TBHQ,高于维生素C。     

ESR study on the structure-antioxidant activity relationship of tea catechins and their epimers

The purpose of this study is to examine the relationship between the free radical scavenging activities and the chemical structures of tea catechins ((-)-epigallocatechin gallate (EGCG), (-)-epigallocatechin (EGC) and (-)-epicatechin (EC)) and their corresponding epimers ((-)-gallocatechin gallate (GCG), (-)-gallocatechin (GC) and (+)-catechin ((+)-C)). With electron spin resonance (ESR) we investigated their scavenging effects on superoxide anions (O-.2) generated in the irradiated riboflavin system, singlet oxygen(1O2) generated in the photoradiation-hemoporphyrin system, the free radicals generated from 2,2'-azobis(2-amidinopropane)hydrochloride (AAPH) and 1, 1-diphenyl-2-picrylhydrazyl (DPPH) radical. The results showed that the scavenging effects of galloylated catechins (EGCG and GCG) on the four free radicals were stronger than those of nongalloylated catechins (EGC, GC, EC, (+)-C), and the scavenging effects of EGC and GC were stronger than those of EC and (+)-C. Thus, it is suggested that the presence of the gallate group at the 3 position plays the most important role in their free radical-scavenging abilities and an additional insertion of the hydroxyl group at the 5' position in the B ring also contributes to their scavenging activities. Moreover, the corresponding phenoxyl radicals formed after the reaction with O-.2 were trapped by DMPO and the ESR spectra of DMPO/phenoxyl radical adducts were observed (aN=15.6 G and aHbeta=21.5 G). No significant differences were found between the scavenging effects of the catechins and their epimers when their concentrations were high. However, significant differences were observed at relatively low concentrations, and the lower their concentrations, the higher the differences. The scavenging abilities of GCG, GC and (+)-C were stronger than those of their corresponding epimers (EGCG, EGC and EC). The differences between their sterical structures played a more important role in their abilities to scavenge large free radicals, such as the free radicals generated from AAPH and the DPPH radical, than to scavenge small free radicals, such as O-.2 and 1O2, especially in the case with EGCG and GCG with more bulky steric hindrance.     

Distinct effects of tea catechins on 6-hydroxydopamine-induced apoptosis in PC12 cells.

Green tea polyphenols have aroused considerable attention in recent years for preventing oxidative stress related diseases including cancer, cardiovascular disease, and degenerative disease. Neurodegenerative diseases are cellular redox status dysfunction related diseases. The present study investigated the different effects of the five main components of green tea polyphenols on 6-hydroxydopamine (6-OHDA)-induced apoptosis in PC12 cells, the in vitro model of Parkinson's disease (PD). When the cells were treated with five catechins respectively for 30 min before exposure to 6-OHDA, (-)-epigallocatechins gallate (EGCG) and (-)-epicatechin gallate (ECG) in 50-200 microM had obvious concentration-dependent protective effects on cell viability, while (-)-epicatechin (EC), (+)-catechin ((+)-C), and (-)-epigallocatechin (EGC) had almost no protective effects. The five catechins also showed the same pattern described above of the different effects against 6-OHDA-induced cell apoptotic characteristics as analyzed by cell viability, fluorescence microscopy, flow cytometry, and DNA fragment electrophoresis methods. The present results indicated that 200 microM EGCG or ECG led to significant inhibition against typical apoptotic characteristics of PC12 cells, while other catechins had little protective effect against 6-OHDA-induced cell death. Therefore, the classified protective effects of the five catechins were in the order ECG> or = EGCG>EC> or = (+)-C>EGC. The antiapoptotic activities appear to be structurally related to the 3-gallate group of green tea polyphenols. The present data indicate that EGCG and ECG might be potent neuroprotective agents for PD.     

Antioxidative effects of green tea polyphenols on free radical initiated and photosensitized peroxidation of human low density lipoprotein

Antioxidative effects of the main polyphenolic components extracted from green tea leaves, i.e. (-)-epicatechin (EC), (-)-epigallocatechin (EGC), (-)-epicatechin gallate (ECG), (-)-epigallocatechin gallate (EGCG) and gallic acid (GA), against free radical initiated peroxidation of human low density lipoprotein (LDL) were studied. The peroxidation was initiated either thermally by a water-soluble initiator 2,2'-azobis(2-amidinopropane hydrochloride) (AAPH), or photochemically by a triplet sensitizer benzophenone (BP). The reaction kinetics was monitored by the uptake of oxygen and the depletion of alpha-tocopherol (TOH) presented in the native LDL. Kinetic analysis of the antioxidation process demonstrates that these green tea polyphenols are effective antioxidants against both AAPH-initiated and BP-photosensitized LDL peroxidation. The antioxidative action of the green tea polyphenols includes trapping the initiating and/or propagating peroxyl radicals with the activity sequence EC>EGCG>ECG>EGC>GA for the AAPH initiated peroxidation, and reducing the alpha-tocopheroxyl radical to regenerate alpha-tocopherol with the activity sequence of ECG>EC>EGCG>EGC>GA and ECG>EGCG>GA>EC>EGC for the AAPH-initiated and BP-photosensitized peroxidations respectively.     

Complex effects of different green tea catechins on human platelets

Epigallocatechin gallate (EGCG), a major component of green tea, has been previously shown to inhibit platelet aggregation. The effects of other green tea catechins on platelet function are not known. Pre-incubation with EGCG concentration-dependently inhibited thrombin-induced aggregation and phosphorylation of p38 mitogen-activated protein kinase and extracellular signal-regulated kinases-1/2. In contrast EGCG stimulated tyrosine phosphorylation of platelet proteins, including Syk and SLP-76 but inhibited phosphorylation of focal adhesion kinase. Other catechins did not inhibit platelet aggregation. Interestingly, when EGCG was added to stirred platelets, a tyrosine kinase-dependent stimulation of platelet aggregation was observed. The two other catechins containing a galloyl group in the 3' position (catechin gallate, epicatechin gallate) also stimulated platelet aggregation, while catechins without a galloyl group (catechin, epicatechin) or the catechin with a galloyl group in the 2' position (epigallocatechin) did not.     

Electron-transfer capacity of catechin derivatives and influence on the cell cycle and apoptosis in HT29 cells

Galloylated and nongalloylated catechin conjugates with cysteine derivatives have been synthesized and evaluated for their capacity to scavenge free radicals and to influence crucial functions (cell cycle, apoptosis) in HT29 colon carcinoma cells. We show that the nonphenolic part of the molecule modified the capacity of catechins to donate hydrogen atoms and to transfer electrons to free radicals. Nongalloylated derivatives did not significantly influence either the cell cycle or apoptosis. Among the galloylated species, 4beta-[S-(O-ethyl-cysteinyl)]epicatechin 3-O-gallate, which showed a high electron-transfer capacity (5 e- per molecule), arrested the cell cycle and induced apoptosis as expected for galloylated catechins such as tea (-)-epigallocatechin 3-O-gallate. 4beta-[S-(N-Acetyl-O-methyl-cysteinyl)]epicatechin 3-O-gallate, which showed the highest hydrogen-donating capacity (10 H per molecule) while keeping the electron-transfer capacity low (2.9 e- per molecule), did not trigger any significant apoptosis. The gallate moiety did not appear to be sufficient for the pro-apoptotic effect of the catechin derivatives in HT29 cells. Instead, a high electron-transfer capacity is more likely to be behind this effect. The use of stable radicals sensitive exclusively to electron transfer may help to design molecules with either preventive scavenging action (high hydrogen donation, low electron transfer) or therapeutic pro-apoptotic activity (high electron transfer).     

Investigate the Binding of Catechins to Trypsin Using Docking and Molecular Dynamics Simulation

To explore the inhibitory mechanism of catechins for digestive enzymes, we investigated the binding mode of catechins to a typical digestive enzyme-trypsin and analyzed the structure-activity relationship of catechins, using an integration of molecular docking, molecular dynamics simulation and binding free energy calculation. We found that catechins with different structures bound to a conservative pocket S1 of trypsin, which is comprised of residues 189–195, 214–220 and 225–228. In the trypsin-catechin complexes, Asp189 by forming strong hydrogen bonding, and Gln192, Trp215 and Gly216 through hydrophobic interactions, all significantly contribute to the binding of catechins. The number and the position of hydroxyl and aromatic groups, the structure of stereoisomers, and the orientation of catechins in the binding pocket S1 of trypsin all affect the binding affinity. The binding affinity is in the order of Epigallocatechin gallate (EGCG) > Epicatechin gallate (ECG) > Epicatechin (EC) > Epigallocatechin (EGC), and 2R-3R EGCG shows the strongest binding affinity out of other stereoisomers. Meanwhile, the synergic conformational changes of residues and catechins were also analyzed. These findings will be helpful in understanding the knowledge of interactions between catechins and trypsin and referable for the design of novel polyphenol based functional food and nutriceutical formulas.     

Radical scavenging activity of tea catechins and their related compounds

(-)-Epigallocatechin gallate was found to be the most effective scavenger among tea catechins for the superoxide anion, hydroxyl radical, and 1,1-diphenyl-3-picrylhydrazyl radical. Examination of the scavenging effects of tea catechins and their glucosides on superoxide anion showed that the presence of at least an ortho-dihydroxyl group in the B ring and a galloyl moiety at the 3 position was important in maintaining the effectiveness of the radical scavenging ability. Stoichiometric factors of tea catechins were estimated to be 2 for (+)-catechin and (-)-epicatechin, 5 for (-)-epigallocatechin, 7 for (-)-epicatechin gallate, and 10 for (-)-epigallocatechin gallate.     

Structure-activity relationship of the tocopherol-regeneration reaction by catechins

The reaction rates (k(r)) of 5,7-diisopropyl-tocopheroxyl radical (Toc) with catechins (epicatechin (EC), epicatechin gallate (ECG), epigallocatechin (EGC), epigallocatechin gallate (EGCG)) and related compounds (methyl gallate (MG), 4-methylcatechol (MC), and 5-methoxyresorcinol (MR)) have been measured by stopped-flow spectrophotometer. The k(r) values increased in the order of MR < < MG < EC < MC approximately ECG < EGC < EGCG in ethanol and 2-propanol/H(2)O (5/1, v/v) solutions, indicating that the reactivity of the OH groups in catechins increased in the order of resorcinol A-ring < < gallate G-ring < catechol B-ring < pyrogallol B-ring. The catechins which have lower oxidation potentials show higher reactivities. The rate constants for catechins in micellar solution showed notable pH dependence with one or two peaks around pH 9-11, because of the dissociation of various phenolic hydroxyl protons in catechins. The structure-activity relationship in the free-radical-scavenging reaction by catechins has been clarified by the detailed analyses of the pH dependence of k(r) values. The reaction rates increased remarkably with increasing the anionic character of catechins, that is, the electron-donating capacity of catechins. The mono anion form at catechol B-and resorcinol A-rings and dianion form at pyrogallol B-and gallate G-rings show the highest activity for free-radical-scavenging. It was found that catechins (EC, ECG, EGC, and EGCG) have activity similar to or higher than that of vitamin C in vitamin E regeneration at pH 7-12 in micellar solution.     

试论茶多酚清除生物自由基的高效性

从生物自由基出发,本文综述了茶多酚(TP)清除自由基的效能.茶多酚对多种自由基具有卓越的清除特性,并明显优于其它抗氧化剂,但茶多酚浓度、体系pH值、自由基类型、儿茶素组成与结构对其清除效能有较大影响.     

Dietary gallate esters of tea catechins reduce deposition of visceral fat, hepatic triacylglycerol, and activities of hepatic enzymes related to fatty acid synthesis in rats

Tea catechins, rich in (-)-epigallocatechin gallate and (-)-epicatechin gallate, or heat-treated tea catechins in which about 50% of the (-)-epigallocatechin gallate and (-)-epicatechin gallate in tea catechins was epimerized to (-)-gallocatechin gallate and (-)-catechin gallate, were fed to rats at 1% level for 23 d. Visceral fat deposition and the concentration of hepatic triacylglycerol were significantly lower in the tea catechin and heat-treated tea catechin groups than in the control group. The activities of fatty acid synthase and the malic enzyme in the liver cytosol were significantly lower in the two catechin groups than in the control group. In contrast, the activities of carnitine palmitoyltransferase and acyl-CoA oxidase in the liver homogenate were not significantly different among the three groups. These results suggest that the reduction in activities of enzymes related to hepatic fatty acid synthesis by the feeding of tea catechins or heat-treated tea catechins can cause reductions of hepatic triacylglycerol and possibly of visceral fat deposition.