响应面优化柚皮苷制备根皮乙酰苯-4'-新橘皮糖苷工艺

本文通过碱催化反应使柚皮苷水解为根皮乙酰苯-4'-β-新橘皮糖苷,利用响应面对溶剂、时间、碱浓度、温度、料液比等主要因素进行了优化。实验结果表明,柚皮苷水解为根皮乙酰苯-4'-新橘皮糖苷的最佳工艺条件如下:氢氧化钾为催化剂,水作为溶剂,碱浓度为15%(w/w),料液比为15(v/w),100℃反应2 h。柚皮苷水解为根皮乙酰苯-4'-新橘皮糖苷的产率最高达70%。     

五种金花茶组植物类黄酮成分及其与花色关系

以金花茶、小果金花茶、扶绥金花茶、龙州金花茶和陇瑞金花茶等五种金花茶组植物为试验材料,按照CIE L*a*b*表色系法测量其花色,利用超高效液相色谱-四极杆-飞行时间质谱(UPLC-Q-TOF-MS)联用技术定性定量分析其花中类黄酮成分与含量,运用多元线性回归方法研究花色与类黄酮成分之间的关系。结果表明:5种金花茶组植物花中共检测到8种类黄酮成分,其中天竺葵素-3-O-葡萄糖苷(Pg3G)、木犀草素-7-O-芸香糖苷(Lu7R)、芸香柚皮苷和圣草素为金花茶组植物中首次发现;槲皮素-3-O-葡萄糖苷(Qu3G)、槲皮素-7-O-葡萄糖苷(Qu7G)、槲皮素-3-O-芸香糖苷(Qu3R)和山柰酚-3-O-葡萄糖苷(Km3G)为扶绥金花茶和小果金花茶中首次发现;金花茶花中类黄酮成分总量最高,其次是扶绥金花茶和小果金花茶,陇瑞金花茶和龙州金花茶较低;金花茶和小果金花茶主要类黄酮成分为Qu3G、Qu3R和Pg3G,扶绥金花茶为Qu3G和Qu7G,陇瑞金花茶和龙州金花茶为圣草素和芸香柚皮苷; Qu3G和Qu3R是决定金花茶组植物花瓣呈现黄色的主要成分,圣草素与花瓣红晕显著正相关,Pg3G影响花色鲜艳程度。     

葡萄柚中糖基转移酶对槲皮素及其类似物催化机制探讨

研究了葡萄柚中糖基转移酶(FGT)作为催化酶,以UDP-葡萄糖、UDP-N-乙酰-D-氨基葡萄糖、UDP-甘露糖为糖基供体,槲皮素、柚皮素、柚皮苷为糖基受体,研究糖基化合成情况。通过高效液相、质谱及核磁共振氢谱对产物反应进行检测。确定了槲皮素与UDP-葡萄糖、UDP-N-乙酰-D-氨基葡萄糖可以发生糖基化反应,生成的槲皮素糖苷分子量分别为464、505。根据核磁结果鉴定,所得到的产物结构分别为槲皮素-3-O-β-D葡萄糖苷、槲皮素3-O-β-D-N-乙酰-D-氨基葡萄糖苷。以FGT酶为催化酶,对槲皮素进行了糖基化修饰。     

崇左金花茶花朵和叶片类黄酮UPLC-Q-TOF-MS分析

以崇左金花茶（Camellia chuangtsoensis）为材料,利用超高效液相色谱-四极杆-飞行时间质谱（UPLC-Q-TOF-MS）联用技术定性定量分析其花朵（花瓣、雄蕊）和叶片（老叶、新叶）中类黄酮成分与含量。结果表明,崇左金花茶中共检测到14种类黄酮成分,木犀草素、木犀草素-7-O-芸香糖苷、槲皮素-3,7-O-二葡萄糖苷、芸香柚皮苷、圣草素和染料木苷为山茶属金花茶组植物中首次发现,其中槲皮素-3,7-O-二葡萄糖苷、芸香柚皮苷、圣草素和染料木苷主要存在于花朵中,木犀草素和木犀草素-7-O-芸香糖苷在花朵中含量高于叶片,雄蕊中高于花瓣;槲皮素-3-O-葡萄糖苷、槲皮素-7-O-葡萄糖苷、槲皮素-3-O-芸香糖苷和山柰酚-3-O-葡萄糖苷为金花茶组植物叶片中首次发现,其叶片中含量远低于花朵,老叶中远低于新叶,雄蕊中远低于花瓣;儿茶素和表儿茶素在花朵中含量高于叶片,雄蕊中高于花瓣;槲皮素和山萘酚在花朵和叶片中含量均较低。崇左金花茶花瓣和雄蕊中含量较高的类黄酮为儿茶素类、木犀草素类和槲皮素类,主要是表儿茶素、木犀草素和槲皮素-3-O-葡萄糖苷;叶片中为儿茶素类和木犀草素类,主要是表儿茶素、木犀草素和木犀草素-7-O-芸香糖苷。崇左金花茶花瓣和雄蕊中儿茶素类、木犀草素类及类黄酮总量均高于叶片,且雄蕊高于花瓣;花瓣和雄蕊中槲皮素类远高于叶片,且花瓣中远高于雄蕊。     

晚期糖基化终末产物荧光光谱检测法在糖尿病筛查中的应用研究

主要研究人体皮肤晚期糖基化终末产物(Advanced glycation end products,AGE)荧光光谱的检测方法,并对AGE荧光光谱在糖尿病检测中的应用价值进行评估.利用研制的AGE荧光光谱检测装置,分别对73例受试者前臂内侧皮肤组织中AGE的荧光进行检测.同时,采用酶联免疫吸附法(ELISA)对受试者血...     

不同生长温度对烟草叶片黄酮类化合物含量及其代谢途径的调控

本研究以生长在3个不同温度(均温18.5℃,均温23.5℃,均温28.5℃)人工气候室中的"云烟87"为材料,运用液相色谱质谱联用法、分光光度法和荧光定量PCR法,研究了不同生长温度对烟叶生长发育过程中黄酮类化合物芸香苷和山奈酚-3-O-芸香苷含量、代谢相关酶活性及相关基因表达的影响。结果表明:随着烟草叶片的生长发育,黄酮类化合物代谢相关基因表达呈先降低后升高的趋势,从而调控了黄酮类化合物代谢相关酶活性的变化趋势为先降低后升高,使得黄酮类化合物中的芸香苷含量变化趋势为先降低后升高,山奈酚-3-O-芸香苷的含量则呈逐渐降低的趋势。此外,与生长在较高生长温度(均温23.5℃和均温28.5℃)下的叶片相比,较低生长温度(均温18.5℃)上调了烟草叶片类黄酮代谢相关基因PAL、C4H、4CL、CHS、F3H、F3'H和FLS的基因表达,提高了类黄酮代谢关键酶PAL、C4H、4CL和CHI的活性,促进了黄酮类化合物芸香苷和山奈酚-3-O-芸香苷的积累(p0.05),表明较低的生长温度(均温18.5℃)有利于黄酮类化合物在烟草叶片中的积累。     

HPLC-Q-TOF/MS分析脐橙果实中的类黄酮

采用高效液相色谱分离、串联四极杆-飞行时间质谱正离子模式检测‘红肉脐橙’和‘清家脐橙’果实黄皮层、白皮层、囊衣和汁胞中的主要类黄酮。根据保留时间、精确质荷比、二级质谱以及标准品化合物验证,确定了脐橙不同组织中含量较高的甜橙黄酮、川陈皮素等13种类黄酮。依据峰面积比较相对含量,认为脐橙黄皮层中类黄酮含量丰富,白皮层和囊衣中类黄酮含量次之,汁胞中类黄酮相对含量较少。黄皮层中以甜橙黄酮、川陈皮素和橘皮素等多甲氧基黄酮为主,而白皮层、囊衣和汁胞中的类黄酮以橙皮苷、柚皮苷为主。脐橙相同组织如白皮层、囊衣和汁胞中类黄酮的相对含量在品种间无显著差异,但橙皮苷、3,5,6,7,3',4'-六甲氧基黄酮和橘皮素在两品种的黄皮层中相对含量差异显著。研究结果为进一步研究和综合利用脐橙的活性物质提供了科学依据。     

HPLC-Q—TOF／MS分析脐橙果实中的类黄酮

采用高效液相色谱分离、串联四极杆-飞行时间质谱正离子模式检测‘红肉脐橙’和‘清家脐橙’果实黄皮层、白皮层、囊衣和汁胞中的主要类黄酮。根据保留时间、精确质荷比、二级质谱以及标准品化合物验证,确定了脐橙不同组织中含量较高的甜橙黄酮、川陈皮素等13种类黄酮。依据峰面积比较相对含量,认为脐橙黄皮层中类黄酮含量丰富,白皮层和囊衣中类黄酮含量次之,汁胞中类黄酮相对含量较少。黄皮层中以甜橙黄酮、川陈皮素和橘皮素等多甲氧基黄酮为主,而白皮层、囊衣和汁胞中的类黄酮以橙皮苷、柚皮苷为主。脐橙相同组织如白皮层、囊衣和汁胞中类黄酮的相对含量在品种间无显著差异,但橙皮苷、3,5,6,7,3’,4’-六甲氧基黄酮和橘皮素在两品种的黄皮层中相对含量差异显著。研究结果为进一步研究和综合利用脐橙的活性物质提供了科学依据。     

化橘红果实生长发育过程中类黄酮的动态变化

该研究建立了化橘红(Citrus grandis cv.‘Tomentosa’)果实中4种类黄酮成分的提取分离、HPLC定量和指纹图谱分析方法,利用该方法测定了果实生长发育过程中类黄酮的动态变化。结果表明,用60%乙醇超声辅助提取果实干燥粉末2小时,柚皮苷提取率达到98%以上。用HPLC分离和测定提取物中柚皮苷、野漆树苷、柚皮素和芹菜素的含量及其指纹图谱。结果显示,15–60天不同果龄的果实中类黄酮含量(占果实干重)随着果龄的增大而降低,柚皮苷、野漆树苷、柚皮素和芹菜素分别从52.5%、0.74%、0.57%和0.23%降低至16.1%、0.11%、0.06%和0.03%;每果中类黄酮总量则随着果龄的增加而大幅提高,从15天的0.55 g提高至60天的7.99 g。上述研究结果表明,果龄对化橘红类黄酮含量、产量及药材质量均有很大的影响。该研究为化橘红的工业生产和质量监控提供了重要依据。     

晚期糖基化终产物通过其受体促进内皮细胞分泌IL-8的研究

探讨晚期糖基化终产物(AGE)修饰蛋白对内皮细胞生成白介素8(IL-8)的作用,及晚期糖基化终产物受体(RAGE)在此病理过程中的作用.内皮细胞来自培养的人脐静脉内皮细胞(HUVEC).将内皮细胞与不同浓度的AGE修饰人血清白蛋白(AGE-HSA)在体外共同培养,或以可溶性晚期糖基化终产物受体(sRAGE)对AGE-HSA进行预处理后再与HUVEC共同培养.用蛋白质液相芯片法检测HUVEC培养上清中IL-8水平,并提取细胞RNA,进行RT-PCR反应,检测细胞中IL-8 mRNA的表达水平.结果表明,AGE-HSA以时间和剂量依赖的方式刺激HUVEC生成IL-8,未经修饰的HSA无此作用.AGE-HSA用sRAGE预处理后,刺激HUVEC生成IL-8的作用被抑制,并且此抑制作用呈剂量依赖的方式.AGE-HSA刺激HUVEC使IL-8 mRNA表达增高,未经修饰的HSA无此作用.sRAGE能够阻断AGE-HSA诱导HUVEC表达IL-8mRNA的作用.整个变化趋势与蛋白质水平一致.研究首次证实,AGE-HSA与细胞表面受体RAGE相互作用可刺激内皮细胞分泌IL-8,并上调IL-8 mRNA的表达.这为研究加速型血管病变的发病机制提供了新视角,也为治疗由AGE增多和潴留所引起的病理损害提供了新靶点.     

Inhibition of advanced glycation end product formation on collagen by rutin and its metabolites

Several lines of evidence suggest that rutin, flavonoid in fruits and vegetables, or one of its metabolites may effectively modulate advanced glycation end product (AGE) formation. Following ingestion, rutin forms metabolites that include 3,4-dihydroxyphenylacetic acid (3,4-DHPAA), 3,4-dihydroxytoluene (3,4-DHT), m-hydroxyphenylacetic acid (m-HPAA), 3-methoxy-4-hydroxyphenylacetic acid (homovanillic acid, HVA) and 3,5,7,3',5'-pentahydroxyflavonol (quercetin). We studied the effects of rutin and its metabolites on the formation of AGE biomarkers such as pentosidine, collagen-linked fluorescence, N(epsilon)-carboxymethyllysine (CML) adducts, glucose autoxidation and collagen glycation, using an in vitro model where collagen I was incubated with glucose. Rutin metabolites containing vicinyl dihydroxyl groups, i.e., 3,4-DHT, 3,4-DHPAA and quercetin, inhibited the formation of pentosidine and fluorescent adducts, glucose autoxidation and glycation of collagen I in a dose-dependent manner, whereas non-vicinyl dihydroxyl group-containing metabolites, i.e., HVA and m-HPAA, were much less effective. All five metabolites of rutin effectively inhibited CML formation. In contrast, during the initial stages of glycation and fluorescent AGE product accumulation, only vicinyl hydroxyl group-containing rutin metabolites were effective. These studies demonstrate that rutin and circulating metabolites of rutin can inhibit early glycation product formation, including both fluorescent and nonfluorescent AGEs induced by glucose glycation of collagen I in vitro. These effects likely contribute to the beneficial health effects associated with rutin consumption.     

Paradoxical impact of antioxidants on post-Amadori glycoxidation: Counterintuitive increase in the yields of pentosidine and Nepsilon-carboxymethyllysine using a novel multifunctional pyridoxamine derivative

The inhibition of post-Amadori advanced glycation end product (AGE) formation by three different classes of AGE inhibitors, carbonyl group traps, chelators, and radical-trapping antioxidants, challenge the current paradigms that: 1) AGE inhibitors will not increase the formation of any AGE product, 2) transition metal ions are required for oxidative formation of AGE, and 3) screening AGE inhibitors only in systems containing transition metal ions represents a valid estimate of potential in vivo mechanisms. This work also introduces a novel multifunctional AGE inhibitor, 6-dimethylaminopyridoxamine (dmaPM), designed to function as a combined carbonyl trap, metal ion chelator, and radical-trapping antioxidant. Other AGE inhibitors including pyridoxamine, aminoguanidine, o-phenylenediamine, dipyridoxylamine, and diethylenetriaminepentaacetic acid were also examined. The results during uninterrupted and interrupted ribose glycations show: 1) an unexpected increase in the yield of pentosidine in the presence of radical-trapping phenolic antioxidants such as Trolox and dmaPM, 2) significant formation of Nepsilon-carboxymethyllysine (CML) in the presence of strong chelators and phenolic antioxidants, which implies that there must be nonradical routes to CML, 3) prevention of intermolecular cross-links with radical-trapping inhibitors, and 4) that dmaPM shows excellent inhibition of AGE. Glucose glycations reveal the expected inhibition of pentosidine and CML with all compounds tested, but in a buffer free of trace metal ions the yield of CML in the presence of radical-trapping antioxidants was between the metal ion-free and metal ion-containing controls. Protein molecular weight analyses support the conclusion that Amadori decomposition pathways are constrained in the presence of metal ion chelators and radical traps.     

Modulation of advanced glycation endproduct synthesis by kynurenines in human lens proteins

Human lens proteins (HLP) become chemically modified by kynurenines and advanced glycation end products (AGEs) during aging and cataractogenesis. We investigated the effects of kynurenines on AGE synthesis in HLP. We found that incubation with 5 mM ribose or 5 mM ascorbate produced significant quantities of pentosidine, and this was further enhanced in the presence of two different kynurenines (200–500 µM): N-formylkynurenine (Nfk) and kynurenine (Kyn). Another related compound, 3-hydroxykynurenine (3OH-Kyn), had disparate effects; low concentrations (10–200 µM) promoted pentosidine synthesis, but high concentrations (200–500 µM) inhibited it. 3OH-Kyn showed similar effects on pentosidine synthesis from Amadori-enriched HLP or ribated lysine. Chelex-100 treatment of phosphate buffer reduced pentosidine synthesis from Amadori-enriched HLP by ∼ 90%, but it did not inhibit the stimulating effect of 3OH-Kyn and EDTA. 3OH-Kyn (100–500 μM) spontaneously produced copious amounts of H₂O₂ (10–25 μM), but externally added H₂O₂ had only a mild stimulating effect on pentosidine but had no effect on N^ε-carboxymethyl lysine (CML) synthesis in HLP from ribose and ascorbate. Further, human lens epithelial cells incubated with ribose and 3OH-Kyn showed higher intracellular pentosidine than cells incubated with ribose alone. CML synthesis from glycating agents was inhibited 30 to 50% by 3OH-Kyn at concentrations of 100–500 μM. Argpyrimidine synthesis from 5 mM methylglyoxal was slightly inhibited by all kynurenines at concentrations of 100–500 μM. These results suggest that AGE synthesis in HLP is modulated by kynurenines, and such effects indicate a mode of interplay between kynurenines and carbohydrates important for AGE formation during lens aging and cataract formation.     

Detection of noncarboxymethyllysine and carboxymethyllysine advanced glycation end products (AGE) in serum of diabetic patients.

BACKGROUND: The advanced stage of the Maillard reaction, which leads to the formation of advanced glycation end products (AGE), plays an important role in the pathogenesis of angiopathy in diabetic patients and in the aging process. N(epsilon)-(carboxymethyl)lysine (CML) is thought to be an important epitope for many of currently available AGE antibodies. However, recent findings have indicated that a major source of CML may be by pathways other than glycation. A distinction between CML and non-CML AGE may increase our understanding of AGE formation in vivo. In the present study, we prepared antibodies directed against CML and non-CML AGE. MATERIALS AND METHODS: AGE-rabbit serum albumin prepared by 4, 8, and 12 weeks of incubation with glucose was used to immunize rabbits, and a high-titer AGE-specific antiserum was obtained without affinity for the carrier protein. To separate CML and non-CML AGE antibodies, the anti-AGE antiserum was subjected to affinity chromatography on a column coupled with AGE-BSA and CML-BSA. Two different antibodies were obtained, one reacting specifically with CML and the other reacting with non-CML AGE. Circulating levels of CML and non-CML AGE were measured in 66 type 2 diabetic patients without uremia by means of the competitive ELISA. Size distribution and clearance by hemodialysis detected by non-CML AGE and CML were assessed in serum from diabetic patients on hemodialysis. RESULTS: The serum non-CML AGE level in type 2 diabetic patients was significantly correlated with the mean fasting blood glucose level over the previous 2 months (r = 0.498, p < 0.0001) or the previous 1 month (r = 0.446, p = 0. 0002) and with HbA(1c) (r = 0.375, p = 0.0019), but the CML AGE level was not correlated with these clinical parameters. The CML and non-CML AGE were detected as four peaks with apparent molecular weights of 200, 65, 1.15, and 0.85 kD. The hemodialysis treatment did not affect the high-molecular-weight protein fractions. Although the low-molecular-weight peptide fractions (absorbance at 280 nm and fluorescence) were decreased by hemodialysis, there was no difference before and after dialysis in the non-CML AGE- and CML-peptide fractions (1.15 and 0.85 kD fractions). CONCLUSIONS: We propose that both CML and non-CML AGE are present in the blood and that non-CML AGE rather than CML AGE should be more closely evaluated when investigating the pathophysiology of AGE-related diseases.     

Nepsilon-(Carboxymethyl)lysine and 3-DG-imidazolone are major AGE structures in protein modification by 3-deoxyglucosone

The levels of plasma 3-deoxyglucosone (3-DG) increase under hyperglycemic conditions and are associated with the pathogenesis of diabetic complications because of the high reactivity of 3-DG with proteins to form advanced glycation end products (AGE). To investigate potential markers for 3-DG-mediated protein modification in vitro and in vivo, we compared the yield of several 3-DG-derived AGE structures by immunochemical analysis and HPLC and measured their localization in human atherosclerotic lesions. When BSA was incubated with 3-DG at 37 degrees C for up to 4 wk, the amounts of N(epsilon)-(carboxymethyl)lysine (CML) and 3-DG-imidazolone steeply increased with incubation time, whereas the levels of pyrraline and pentosidine increased slightly by day 28. In contrast, significant amounts of pyrraline and pentosidine were also observed when BSA was incubated with 3-DG at 60 degrees C to enhance AGE-formation. In atherosclerotic lesions, CML and 3-DG-imidazolone were found intracellularly in the cytoplasm of most foam cells and extracellularly in the atheromatous core. A weak-positive immunoreaction with pyrraline was found in the extracellular matrix and a few foam cells in aortic intima with atherosclerotic lesions. Our results provide the first evidence that CML and 3-DG-imidazolone are major AGE structures in 3-DG-modified proteins, and that 3-DG-imidazolone provides a better marker for protein modification by 3-DG than pyrraline.     

Natural compounds containing a catechol group enhance the formation of Nε-(carboxymethyl)lysine of the Maillard reaction

Inhibition of advanced glycation end-product (AGE) formation is a potential strategy for the prevention of clinical diabetes complications. Screening for new AGE inhibitors revealed several natural compounds that inhibited the formation of N(ε)-(carboxymethyl)lysine (CML), a major antigenic AGE structure, whereas natural compounds containing a catechol group, such as gallic acid and epicatechin, significantly enhanced CML formation. A similar enhancing effect was also observed by culturing THP-1 macrophages in the presence of catechol compounds. Although 4-methylcatechol significantly enhanced CML formation from glycated HSA (gHSA), a model for Amadori proteins, analogues of catechol such as 5-methylresorcinol and methylhydroquinone showed no enhancing effect. Even though 1mM 4-methylcatechol, epicatechin, and gallic acid significantly enhanced CML formation from gHSA, it was significantly inhibited by decreasing their concentration. The enhancing effect of 1mM catechol compounds was inhibited in the presence of the glutathione peroxidase system, thus demonstrating that hydrogen peroxide generated from catechol compounds plays an important role in the enhancement of CML formation. Furthermore, administration of 500mg/kg/day epicatechin to STZ-induced diabetic mice for 45days enhanced CML accumulation at the surface area of gastric epithelial cells in the stomach. This study provides the first evidence that high amounts of catechol-containing structures enhance oxidative stress, thus leading to enhanced CML formation, and this phenomenon may explain the paradoxical effect that some flavonoids have on redox status.     

Inhibition of advanced glycation by flavonoids. A nutritional implication for preventing diabetes complications?

Advanced glycation of collagens contributes to development of micro- and macrovascular complications in diabetes. Since flavonoids are potent natural antioxidants, it was interesting to examine their effect on the formation of a cross-linking advanced glycation endproduct, pentosidine, in collagen incubated with glucose. Monomeric flavonoids (25 and 250 microM) markedly reduced pentosidine/hydroxyproline values in a concentration- and structure-dependent manner. Procyanidin oligomers from grape seed were more active than pine bark procyanidin oligomers. Oligomers are known to be cleaved into monomers in the gastric milieu and monomeric flavonoids to be absorbed and recovered at micromolar concentrations (with a long plasmatic half-life) in extracellular fluids, in contact with collagens. In conclusion, flavonoids are very potent inhibitors of pentosidine formation in collagens, active at micromolar concentrations; these concentrations might be achieved in plasma of diabetic patients after oral intake of flavonoids.     

A sensitive and specific HPLC method for the determination of total pentosidine concentration in plasma

Pentosidine is an advanced glycation end-product (AGE) appearing when arginine and lysine residues in proteins are cross-linked with carbonyl derivatives. This paper presents an improved method for the synthesis of pentosidine and reversed-phase chromatography of this substance with fluorometric detection that enables sensitive (0.01 pmol/mg protein) and specific determination of pentosidine in plasma. Separation is done twice on the same C(18) Vydac 218TP54 column, first with trifluoroacetic acid and next with heptafluorobutyric acid as ion pair. The inter-day coefficient of variation is 6.4% at pentosidine concentration in plasma of 25 pmol/mg protein and 8% at 1.7 pmol/mg protein. Spectral properties of pentosidine exploited during identification of the substance with UV absorption and fluorescence detectors are described. Maximum of absorbance was observed at 325 nm, maximum fluorescence at lambda(ex)/lambda(em)=330/373 nm. The method may prove useful for the study of processes associated with generation and accumulation of pentosidine in the body as a marker of AGE production in healthy subjects and patients with chronic renal failure.     

The combined effect of acetylation and glycation on the chaperone and anti-apoptotic functions of human α-crystallin

N^ε-acetylation occurs on select lysine residues in α-crystallin of the human lens and alters its chaperone function. In this study, we investigated the effect of N^ε-acetylation on advanced glycation end product (AGE) formation and consequences of the combined N^ε-acetylation and AGE formation on the function of α-crystallin. Immunoprecipitation experiments revealed that N^ε-acetylation of lysine residues and AGE formation co-occurs in both αA- and αB-crystallin of the human lens. Prior acetylation of αA- and αB-crystallin with acetic anhydride (Ac₂O) before glycation with methylglyoxal (MGO) resulted in significant inhibition of the synthesis of two AGEs, hydroimidazolone (HI) and argpyrimidine. Similarly, synthesis of ascorbate-derived AGEs, pentosidine and N^ε-carboxymethyl lysine (CML), was inhibited in both proteins by prior acetylation. In all cases, inhibition of AGE synthesis was positively related to the degree of acetylation. While prior acetylation further increased the chaperone activity of MGO-glycated αA-crystallin, it inhibited the loss of chaperone activity by ascorbate-glycation in both proteins. BioPORTER-mediated transfer of αA- and αB-crystallin into CHO cells resulted in significant protection against hyperthermia-induced apoptosis. This effect was enhanced in acetylated and MGO-modified αA- and αB-crystallin. Caspase-3 activity was reduced in α-crystallin transferred cells. Glycation of acetylated proteins with either MGO or ascorbate produced no significant change in the anti-apoptotic function. Collectively, these data demonstrate that lysine acetylation and AGE formation can occur concurrently in α-crystallin of human lens, and that lysine acetylation improves anti-apoptotic function of α-crystallin and prevents ascorbate-mediated loss of chaperone function.     

Tomato paste fraction inhibiting the formation of advanced glycation end-products

A water-soluble and low-molecular-weight fraction (SB) was obtained from tomato paste. The effects of SB on the formation of advanced glycation end-products (AGE) in protein glycation were studied by the methods of specific fluorescence, ELISA and a Western blot analysis, using the anti-AGE antibody after incubating protein with sugar. The results suggest that SB had strong inhibitory activity, in comparison with aminoguanidine as a positive control, and that the inhibitory mechanism of SB differed from that of aminoguanidine to involve trapping of reactive dicarbonyl intermediates in the early stage of glycation. SB contained an antioxidant, rutin, which showed potent inhibitory activity. The results also suggest that rutin chiefly contributed to inhibiting the formation of AGE, and that other compounds in SB may also have been related to the activity.