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Enzymatic Synthesis and Antioxidant Properties of Poly (rutin)聚（芦丁）的酶促合成与抗氧化性质Motoichi Kurisawa, Joo Eun Chung, Hiroshi Uyama,* and Shiro Kobayashi*Abstract 摘要Rutin, quercetin-3-rutinoside, is one of the most famous glycosides of flavonoid and widely present in many plants. In this study, we performed an oxidative polymerization of rutin using Myceliophthora laccase as catalyst in a mixture of methanol and buffer to produce a flavonoid polymer and evaluated antioxidant properties of the resultant polymer. Under selected conditions , the polymer with molecular weight of several thousands was obtained in good yields. The resulting polymer was readily soluble in water, DMF, and DMSO, although rutin monomer showed very low water solubility. UV measurement showed that the polymer had broad transition peaks around 255 and 350 nm in water, which were red-shifted in an alkaline solution. Electron spin resonance (ESR) measurement showed the presence of a radical in the polymer. The polymer showed greatly improved superoxide scavenging activity and inhibition effects on human low-density lipoprotein (LDL) oxidation initiated by 2,2-azobis(2-amidino- propane) dihydrochloride (AAPH), compared with the rutin monomer. The polymer also protected endothelial cells from oxidative injury induced by AAPH as a radical generator with a much greater effect than the rutin monomer. 芦丁，槲皮素-3-芸香糖苷，是最著名的黄酮类糖苷之一，广泛存在于很多植物中。在本研究中，我们用毁丝酶属漆酶做催化剂，在甲醇和缓冲剂的混合物中进行了芦丁的氧化聚合，得到了一个黄酮类聚合物并评价了生成聚合物的抗氧化性质。在选定的条件下，分子量为几千的聚合物的产率较好。尽管芦丁单体显示出很低的水溶性，但其生成的聚合物可以很好地溶于水，DMF及DMSO。UV测量显示，该聚合物溶于水在255nm和350nm处有较宽的跃迁峰，这是在碱性溶液中红移的结果。电子自旋共振测量显示，该聚合物中有一个自由基。与芦丁单体相比，该聚合物通过2,2-偶氮(2-甲基丙基脒)二盐酸盐（AAPH）的诱导，显示出较强的超氧化物清除活性，和较强的对人体低密度脂蛋白的氧化的抑制作用。AAPH作为自由基发生器，可诱导该聚合物保护内皮细胞免受氧化伤害，比芦丁单体的作用更强。Introduction 简介Recent interest in flavonoids has increased greatly due to their biological and pharmacological activity including antioxidant, anticarcinogenic, probiotic, antimicrobial, and antiinflammatory properties.1 The flavonoids consist of a large group of low molecular weight polyphenolic substances, naturally occurring in fruits, vegetables, tea, and wine, and are an integral part of the human diet. 最近,由于黄酮类的生物和药理活性，包括抗氧化，抗癌，益生素，抗菌和抗感染等活性，人们对类黄酮的研究兴趣大大增加。黄酮类化合物由大量低分子量的多酚类物质组成，天然存在于水果，蔬菜，茶，酒中，并且是人类饮食中不可或缺的一部分。Rutin (1) is one of the most commonly found flavonol glycosides identified as vitamin P with quercetin and hesperidin (Chart 1) and widely present in many plants, especially the buckwheat plant. Rutin has been reported to have clinically relevant functions, including antioxidant, antihypertensive, antiinflammatory, and antihemorrhagic activity, the strengthing of the capillaries of blood vessels and the regulation of the capillary permeability, and the stabilization of platelets.2 These properties are potentially beneficial in preventing diseases and protecting the stability of the genome. Many of these activities have been related to its antioxidant actions. 芦丁是最常见的黄酮醇糖苷之一，鉴定含维生素P,与槲皮素，橙皮苷广泛存在于很多植物中，特别是荞麦。芦丁已经被报道有临床相关功能，包括抗氧化，抗高血压，抗感染，加固血管的毛细血管和调节毛细血管通透性，稳定血小板。这些性质有助于预防疾病和保护基因组的稳定性。大部分这些作用与它的抗氧化作用有关。Chart 1. Structure of Rutin (1) and Water-Soluble Rutin Derivative (2) 图1 芦丁的结构和水溶性的芦丁衍生物In general, the activities of flavonoids are known to be limited for only few hours in a body, although the metabolism has not been established. In addition, several flavonoids have been shown to act as prooxidants and generate reactive oxygen species, such as hydrogen peroxide. In contrast, a relatively high molecular fraction of extracted flavonoids has been reported to exhibit enhanced physiological properties, such as antioxidant and anticarcinogenic activity, and a relatively longer circulation time in vivo.3 High molecular weight plant polyphenols have also been reported to show no prooxidant effects.4 Many investigations have explored the antioxidant effects of low molecular weight flavonoids, but few have considered polymeric flavonoids. We have designed various polymerized flavonoids or polymeric flavonoid conjugates, in consideration of extension of the amplification of physiological properties of the flavonoids. We reported recently that poly(catechin) as one of the strategic molecular designs was synthesized by the enzyme-catalyzed oxidative coupling using horseradish peroxidase as a catalyst and exhibited great improvement in radical scavenging activity, protection effects against low-density lipoprotein (LDL) oxidation, and inhibition effects on xanthine oxidase activity, compared with a catechin monomer.5 一般而言，已知的黄酮类化合物的作用是有限的，在体内仅维持几个小时，尽管其新陈代谢还没有确定。此外，一些黄酮类显示出促氧化剂的性质，能产生活性氧，如过氧化氢。相反，分子量相对较大的黄酮类提取物的碎片被报道能提高生理活性，像抗氧化和抗癌，且在体内循环的时间较长。分子量大的多酚类植物也被报道不显示促氧化作用。大量的研究报道了低分子量的黄酮类的抗氧化作用，但是很少有关注黄酮类聚合物的。考虑到黄酮类的生理性质的详述的范围，我们设计了各种黄酮类的聚合物及黄酮类共轭物的聚合物。最近我们报道了儿茶酚的聚合物作为战略性设计的分子之一，它是通过辣根过氧化物酶催化的酶促氧化耦合反应合成的，与儿茶酚单体相比，它在自由基清除活性，保护低密度脂蛋白不被氧化及抑制黄嘌呤氧化酶的活性等方面显示出很大的提高。In this study, we synthesized poly(rutin)s by the enzyme-catalyzed oxidative coupling using laccase as a catalyst to amplify the antioxidant activity of rutin and investigated their scavenging activity against reactive oxygen species and protection effects from peroxidation of LDL and from oxidative injury of endothelial cells. 在本研究中，我们以漆酶为催化剂，通过酶促氧化耦合反应合成了聚（芦丁），以详述芦丁的抗氧化活性，并研究了它们对活性氧的清除作用，对LDL的氧化的保护作用及对内皮细胞的氧化损伤的保护作用。Experimental Section 实验部分Materials. Rutin (1) was purchased from Tokyo Kasei Co. The purity of rutin is above 98%. A rutin derivative (2, -G-rutin PS) and laccase (5.7 107 units) derived from Myceliophthora were kindly donated by Toyo Sugar Refining Co., Ltd. and Novozymes Japan Ltd., respectively. Low-density lipoprotein (LDL) from human plasma was purchased from Sigma. Xanthine, xanthine oxidase (from butter milk), 2,2-azobis(2-amidinopropane)dihydrochloride (AAPH), and ethylenediamine tetraacetic acid (EDTA) were obtained from Wako Pure Chemical Industries, Japan. 2-Methyl-6-p-methoxyphenyl ethenyl imidazo pyridine (MPEC) was purchased from Atto Co., Ltd., Japan. Diphenyl-1-pyrenylphosphine (DPPP) was purchased form Dojindo, Japan. Bovine aortic endothelial cells were purchased from Dainippon Pharmaceutical Co., Ltd., Japan. Alamar blue was purchased from Trek Diagnostic Systems Ltd., U.K. Other reagents and solvents are commercially available and used as received. 材料 芦丁购于Tokyo Kasei公司，其纯度大于98%。芦丁衍生物(2, -G-rutin PS)和来自毁丝属漆酶衍生物分别是由日本Toyo Sugar和Novozymes公司捐赠的。来自人血浆的低密度脂蛋白购于Sigma公司。黄嘌呤，黄嘌呤氧化酶，2,2-偶氮二异丁基脒二盐酸盐AAPH，乙二胺四乙酸EDTA ,购于日本光纯药工业株式会社。2-甲基-6-对甲氧基乙烯基咪唑吡啶(MPEC)购于Atto公司，二苯基- 1 -芘磷化氢DPPP购于日本Dojindo公司，牛主动脉内皮细胞购于大日本制药有限公司，alamar blue购于Trek Diagnostic Systems公司，其他试剂和溶液都是标准的商业途径可得到的。Laccase-Catalyzed Synthesis and Characterization of Poly(rutin)s. In a 50 mL flask, rutin (0.20 g) was dissolved in a mixture of methanol and buffer (20 mL). Laccase solution was added to the mixture, followed by gentle stirring for 24 h at room temperature under air. The reaction solution was dialyzed (cut off molecular weight 1 103) in water. The remaining solution was lyophilized to give the polymer. Polymer structure was analyzed by NMR (Bruker DPX400), FT-IR (Perkin-Elmer Spectrum One equipped with universal ATR sampling accessory), UVvisible (Hitachi U-2001), fluorescence (Hitachi F-2500), and electron spin resonance (ESR) (JEOL JES-TE100) spectrometers. UVvisible spectrum of rutin (1) was measured after dilution of rutin/methanol stock solution with a quite excess amount of water. Rutin is insoluble in water, but rutin that was once dissolved in methanol does not form any precipitate when the solvent is exchanged into water. Molecular weight was estimated by size exclusion chromatography (SEC, Tosoh GPC-8020 equipped with RI-8020 detector). The SEC analysis was performed with TSKgel 3000 column and DMF containing 0.10 M LiCl eluent at a flow rate of 0.50 mL/min at 60 C. The calibration curves for SEC analysis were obtained using polystyrene standards. 漆酶催化合成和聚（芦丁）的鉴定 取一个50ml圆底烧瓶，将0.2克芦丁溶于20ml甲醇和缓冲液中。往化合物中加入漆酶溶液，室温下暴露于空气中搅拌24小时。反应液在水中透析（截留分子量为103），剩余溶液冻干得到聚合物。聚合物的结构用NMR,傅里叶变换红外色谱，紫外可见光谱，荧光，电子自旋共振光谱分析。芦丁的紫外可见光谱是在用大量水稀释芦丁/甲醇母液之后测量的。芦丁不溶于水，但是一旦溶于甲醇后，再把溶剂换成水时不会生成任何沉淀物。通过分子排阻色谱法估计分子量。分子排阻色谱分析是通过TSKgel 3000柱完成的，含有0.1摩尔LiCl的DMF作为洗脱剂，流速0.5ml/min,温度60度。分子排阻色谱的标准曲线是通过标准的聚苯乙烯获得的。Superoxide Scavenging Activity. Superoxide anion was generated by xanthine/xanthine oxidase (XO) and measured by chemiluminescent superoxide probe method.6 The chemiluminescence (CL) intensity of MPEC triggered by superoxide anion was measured in a 100 mM potassium phosphate buffer solution (pH 7.5) containing 0.05 mM EDTA, 0.04 unitmL-1 of XO, MPEC, and a test sample. Light emission was started by the addition of 0.3 mM of xanthine. CL spectra were monitored for 30 s using a Corona microplate photoncounter, MTP-700CL (Corona Electric Ltd. Japan). Superoxide anion scavenging activity was calculated according to the following formula. where CLcontrol and CLsample represents chemiluminescent intensity in the absence and presence of samples, respectively. 超氧化物清除活性 超氧化物负离子是由黄嘌呤和黄嘌呤氧化物产生的，并通过化学发光探针方法测量。超氧化物负离子的触发的化学发光强度是在100mM的钾-磷酸缓冲溶液中测量的，缓冲溶液中含有0.05 mM EDTA, 0.04 unitmL-1的黄嘌呤氧化酶, MPEC, 和一个待测样品。加入0.3 mM的黄嘌呤后开始发光。用Corona微孔板光子计数器，MTP-700CL，检测化学发光光谱30秒。根据下面公式估算超氧化物负离子的清除活性。字典 - 查看字典详细内容Determination of LDL Susceptibility to Oxidation. LDL (100 g/mL) was incubated with 200 M (final concentration) of DPPP for 5 min at 37 C under N2 in the dark. The DPPP-labeled LDL was preincubated with samples (070 M) for 1 h at 37 C in the dark. Oxidation of LDL was carried out by further incubation with AAPH (1mM) for 25 h at 37 C. Oxidation of DPPP was measured at the indicated times using a 1420 ARVOSX multilabel counter (Wallac). Wavelengths of excitation and emission were set at 355 and 405 nm, respectively. LDL对氧化剂的敏感性的测定 LDL是在200M的DPPP中，在黑暗中氮气保护下培养5min得到的,温度为37度。DPPP-标记的LDL和样品在37度下的黑暗环境中预培养一个小时。LDL的氧化物进一步在37 度下与AAPH培养25小时。DPPP的氧化物在标明的时间用1420 ARVOSX多功能计数器测量。激发和发射波长分别设在355nm和 405nm 。Cell Culture. Bovine aortic endothelial cells were cultured in Dulbeccos modified Eagles medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 units/mL of penicillin, and 100 g/mL of streptomycin in a humidified atmosphere 5% CO2 incubator. 细胞培养 牛的主动面的内皮细胞是在DMEM培养基中培养的，补充10%的胎牛血清，100单位/ml青霉素,100g/mL链霉素，在含有的CO2湿度5%的培养箱中进行。Evaluation of Protection Effects on Radical-Induced Cytotoxicity. Bovine endothelial cells were seeded at a concentration of 3 105 cells/mL, 200 L/well, in 96-well flat-bottomed microassay plates (Falcon 3072 Co., Becton Dickenson, Flanklin Lakes, NJ) for 24 h before adding sample solutions. A desired amount of sample stock solutions was diluted into DMEM without FBS. The sample solution (190 L) was added into 96-well plates, and preincubated for 1 h at 37 C. AAPH solution (10 L, 200 mM) as a radical generator was added into the well, and the cells were cultured for 48 h at 37 C in a humidified 5% CO2 atmosphere. To evaluate of cell viability, culture media was replaced with RPMI 1640 containing 10% alamar blue, a dye that changes color from blue to red when subjected to reduction by cytochrome c activity, and incubated for 4 h at 37 C.7 Optical absorbance was read at 560 and 600 nm to obtain a dye reduction amount using a 1420 ARVOSX multilabel counter (Wallac). 对自由基诱导的细胞毒素的保护效应的评价 在加入样品之前，牛的内皮细胞以3 105 cells/mL, 200 L/well的浓度，在96号平底微量检测瓶中培养24小时。预得到数量的样品母液的稀释后加到没有胎牛血清的DMEM培养基中。样品溶液加到96号瓶中，37度下培养1小时。AAPH 作为自由基发生器加入瓶中，细胞在湿度5%的CO2环境中培养48小时。为了评价细胞的活性，培养基用含有10% alamar blue 的RPMI 1640代替，当用细胞色素C还原时，染料从蓝色变为红色，在37度培养4小时 。光吸收在560-600nm，用1420 ARVOSX多标签计数器得到染料还原的数量。Results and Discussion 结果和讨论Laccase-Catalyzed Polymerization. Enzymatic oxidation of flavonoid polyphenols is very important in biochemistry because the subsequent coupling reactions are involved in some biosynthetic pathways such as tannin and melanin formation. In the oxidative coupling of catechin by peroxidase or polyphenol oxidase, oligomeric compounds with complicated structure were formed.5,8 漆酶催化聚合作用 黄酮类多酚的酶促反应在生物化学中是非常重要的，因为随后的耦合反应与某些生物合成途径有关，如单宁和黑色素形成。过氧化物酶，多酚氧化酶，低聚物和儿茶酚的氧化耦合形成复杂的结构。Peroxidase was reported to catalyze an oxidation of rutin with use of hydrogen peroxide as an oxidizing agent.9 The reaction monitoring by UVvisible spectroscopy showed the formation of o-quinone intermediate, which may be further polymerized. In a mixture of 1,4-dioxane and buffer, the polymer with molecular weight of 2.3 103 was obtained with the peroxidase catalysis in 30% yield.10 有人报道以过氧化氢为氧化剂，用过氧化物酶催化芦丁的氧化。紫外可见光谱检测反应显示有邻苯醌中间体的形成，可以进一步的聚合。在1,4-二氧杂环乙烷和缓冲溶液的化合物中，用过氧化物酶催化，得到分子量为2.3 103的聚合物，产率为30%。Laccase is a protein containing copper in its active site and uses oxygen as an oxidizing agent. So far, laccase has been used as catalysts for polymerization and curing of phenol derivatives, yielding high-performance polymeric materials.11 In recent years, this laccase-catalyzed polymerization has retained the attention due to an environmentally benign process of polymer production in air without the use of hydrogen peroxide as an oxidizing agent. In this study, we used laccase derived from Myceliophthora as a catalyst, which showed high catalytic activity for the oxidative polymerization of syringic acid.12 Because rutin is readily soluble in methanol but insoluble in water, the polymerization was performed in a mixed solvent of methanol and buffer at room temperature for 24 h. Molecular weight of the polymer was estimated by size exclusion chromatography (SEC) with DMF containing 0.10 M LiCl as eluent. Polymerization results are summarized in Table 1. 漆酶是一种蛋白质，在它的活性位点含有铜离子，并用氧气作为其氧化剂。目前，漆酶已被用作聚合催化剂和苯酚衍生物固化，产生高性能聚合物材料。近年来，由于聚合物的生产过程在空气中进行，没有使用过氧化氢做氧化剂，对环境无害，所以漆酶催化的聚合反应得到了重视。在本研究中，我们用毁丝酶属的漆酶衍生物做催化剂，它对丁香酸的氧化聚合显示出很高的催化活性。因为芦丁易溶于甲醇而不溶于水，所以聚合反应在甲醇和缓冲溶液的混合物中进行，室温下反应24h。聚合物的分子量用排阻色谱法估测，用含有0.10 M LiCl的DMF做洗脱剂。聚合作用的结果如表1所示。In the enzymatic oxidative polymerization of phenol derivatives in an aqueous organic solvent, powdery polymers were often precipitated during the polymerization.13 However, the present polymerization proceeded in the homogeneous phase without any precipitation. After dialysis of the reaction mixture, a brown powdery polymer with molecular weight of several thousands was obtained by lyophilization. In all cases, a unimodal peak was seen in the SEC trace. In an equivolume mixture of methanol and acetate buffer (pH 5), the polymer yield increased as a function of the enzyme amount, whereas the molecular weight did not depend on the enzyme amount (entries 24). The yield and molecular weight of the polymer obtained in the polymerization using the acetate buffer as a cosolvent were relatively close to that in a phosphate buffer of pH 7 (entries 3 and 6). A similar behavior was observed in the laccase-catalyzed polymerization of syringic acid in an aqueous acetone.12 苯酚衍生物在含水的有机溶剂中进行酶氧化聚合反应，反应过程中常常会有粉状聚合物析出。但是，当反应在均相溶剂中进行时就没有任何沉淀。反应得到的混合物透析后，冻干，得到了分子量为几千的棕色粉状聚合物。在所有的实验中，都会在SEC图谱中看到一个单峰。在甲醇和醋酸缓冲溶液(pH 5)中的混合物中，聚合物的产率随着酶的数量增加而增加，而其分子量不随酶数量的变化而变化。用醋酸缓冲溶液做共溶剂的聚合反应得到的聚合物的分子量和产率，与以pH 7的磷酸做共溶剂的很接近。在含水的丙酮中进行的丁香酸的聚合反应中也观察到了相似的情况。In a mixture of methanol and the acetate buffer, the effect of the buffer content was examined (entries 1, 3, and 5). Although the mixed ratio scarcely affected the polymer yield, using 70% buffer afforded the polymer of the highest molecular weight (entry 5). The polymerization also proceeded in an equivolume mixture of methanol and distilled water to produce the polymer in a high yield (entry 7). In entries 3 and 7, quantitative consumption of rutin was confirmed by SEC analysis of the product before dialysis. The monomer shows a very low solubility in water (less than 0.1% at room temperature), whereas the water solubility of the polymer was very high (larger than 5%) (Table 2). The polymer was soluble in N,N-dimethylformamide (DMF) and dimethyl sulfoxide (DMSO) besides water. The solubility of the polymer toward pyridine and methanol was inferior to that of the monomer. 在甲醇和醋酸缓冲溶液的混合物中，我们检验了缓冲溶液含量对反应的影响。虽然混合物的比例几乎不影响聚合物的产率，但用70%的缓冲溶液能得到最大分子量的聚合物。把反应溶剂换成甲醇和蒸馏水也得到高产率的聚合物。在项目3和7中，产品透析前，用SEC分析证实了芦丁的定量消耗。单体显示出很低的水溶性（室温下小于0.1%），而聚合物的水溶性很高（大于5%）。除水之外，聚合物还溶于DMF和DMSO。聚合物在吡啶和甲醇中的溶解性较单体的低。A rutin derivative (2) soluble in water is commercially available, which consists of 2a and 2b (20:80 mol %) (Chart 1). This derivative was also polymerized by the laccase catalyst; the laccase-catalyzed polymerization in an equivolume mixture of methanol and acetate buffer produced the polymer in a high yield (entry 8). The molecular weight of the polymer reached 1 104, which was larger than that from rutin. The polymer from 2 was readily soluble in DMF, DMSO, and water. The polymerization also proceeded in the acetate buffer without use of an organic solvent, although the polymer yield was smaller than that in the aqueous methanol (entry 9). 芦丁衍生物（2）是商业途径可得到的，溶于水，由2a和2b组成。这个衍生物也是漆酶催化聚合产生的；在甲醇和醋酸缓冲溶液中等量的混合物发生聚合反应，得到较高产率的该聚合物。聚合物的分子量达1 104,大于芦丁聚合物的。2的聚合物溶于DMF,DMSO及水。这个聚合反应也是在醋酸缓冲溶液中进行的，没有有机溶剂，尽管聚合物的产率较在含水的甲醇中的低。1H NMR of rutin and poly(rutin) was measured in DMSO-d6. In the spectrum of the polymer, broad peaks were observed at 2.54.0, 4.05.6, and 6.28.5, whereas the monomer has sharp peaks in these regions. Figure 1 shows IR spectra of rutin and poly(rutin) (entry 3). The peak pattern of poly(rutin) was similar to that of rutin, although all of the peaks of poly(rutin) became broader. In the spectrum of poly(rutin), a broad peak centered at 3340 cm-1 due to the vibration of OH linkage of phenolic and hydroxyl groups, a peak at 1650 cm-1 ascribed to the carbonyl vibration, and a peak at 1595 cm-1 ascribed to the CC vibration of aromatic groups were observed. Peaks at 1521, 1122, 971, and 592 cm-1 became smaller, and characteristic peaks did not newly appear. 芦丁和聚(芦丁)的氢谱是在溶剂在DMSO-d6中测量的。在聚合物的波谱中，在 2.54.0, 4.05.6, and 6.28.5出观察到宽峰，而单体在相应的区域为单峰。图1显示了芦丁和芦丁聚合物的红外光谱。尽管芦丁聚合物的所有峰形都变宽