吡喃花色苷类衍生物家族的研究进展

吡喃花色苷是近些年发现于果酒（如红葡萄酒）中的新型花色苷衍生物,是酒体最重要的呈色物质之一。吡喃花色苷家族具有第四个吡喃环的基本特征,是在发酵、陈酿过程中由浆果花色苷与葡萄糖发酵代谢的中间产物及（或）浆果中其他酚类成分经环加合反应形成的一系列天然色素物质。本文将系统介绍吡喃花色苷家族及其第二代衍生物家族的种类、分子结构、形成机制、稳定性与色度特征、抗氧化及抗肿瘤等生物活性。多种不同类型的吡喃花色苷家族不仅是重要呈色物质,而且具有较高的稳定性、良好的色泽特征及较强的功能活性,本文为进一步开展吡喃花色苷类衍生物的结构与其稳定性、色泽和功能性质关系的研究及其在葡萄酒产业和食品加工业中的应用提供有益的参考。     

红葡萄酒花色苷结构和颜色的关系研究进展

花色苷是红葡萄酒颜色的主要物质基础.本文根据花色苷结构,对其进行了分类,即基本花色苷(非酰化花色苷)、酰化花色苷、吡喃花色苷和聚合花色苷;并对其结构和颜色的关系进行了综述讨论,为葡萄酒颜色机理的研究提供参考.     

花色苷分子结构修饰及其生理活性研究进展

花青素是植物界分布最广、种类众多的一类天然色素,是植物的主要呈色物质,在自然界中多以糖苷（花色苷）的形式存在,具有多种重要的生理功能和生物活性。花色苷具有酚羟基,结构稳定性较差,易受外界条件如pH值、温度、光、O2、金属离子和酶等因素的影响,在食品加工中易分解失去原有的鲜艳颜色,限制了花色苷的贮藏和在食品加工领域中的应用。花色苷分子结构修饰可增加其对外界环境和食品添加剂及食品加工中的稳定性,使其在较大pH值范围内呈现良好色泽。本文系统介绍了不同类型（花色苷酰基化、花色苷酯基化及形成吡喃类衍生物）花色苷结构修饰的原料来源、结构特征及其生理活性,为进一步开展花色苷的结构稳定性与生理活性研究及其在食品加工领域中的护色与应用提供参考。     

甲基吡喃花色苷的制备优化及HPLC-MS/MS分析

以葡萄皮红色素提取物为原料,正交实验法研究花色苷在丙酮介质中反应合成甲基吡喃花色苷的最佳条件,探讨了反应物比例、底物浓度、温度、pH等因素对甲基吡喃花色苷生成量的影响,高效液相色谱(HPLC)监测反应过程,并利用高效液相色谱-二极管阵列检测-串联离子阱多级质谱(HPLC-DAD-EI-MS/MS)法对反应产物进行定性和定量分析。结果表明最佳反应条件是:反应物比例(葡萄皮红色素提取物mg∶丙酮mL)15∶1,葡萄皮红色素提取物浓度2.5mg/mL,反应pH3.0、温度45℃。高效液相色谱串联质谱结合多级质谱裂解分析表明,反应主要产物为甲基吡喃锦葵花色苷,反应第9d其得率为49.4%。经分离纯化后的甲基吡喃花色苷特征吸收峰在478nm,与原花色苷比较明显发生蓝移。实验结果为甲基吡喃花色苷的高效制备、功能与理化性质研究提供了一种经济有效的研究方法和物质基础。     

萝卜花色苷的结构研究

红皮白心萝卜的色素提取物经大孔树脂XAD-7HP和葡聚糖凝胶Sephadex LH-20纯化后,结合半制备色谱制备花色苷单体,最后通过LC-MSn和NMR技术手段对萝卜花色苷单体的结构进行分析。通过LC-MSn鉴定出萝卜皮中主要存在四种主要的花色苷,都是以天竺葵色素为苷元,带一个单糖和一个二糖的花色苷衍生物,即以天竺葵-3-槐糖苷-5-葡糖苷为基本结构,带有一个或是两个酰基化的基团,如丙二酸、阿魏酸或香豆酸。经由1H NMR方法,其中三种花色苷的具体结构鉴定为:3-[6-(对香豆酰)-L-葡萄苷(2→1)-葡糖苷]-5-D-吡喃葡糖苷天竺葵素、3-[6-(阿魏酰)-L-葡萄苷(2→1)-葡糖苷]-5-D-吡喃葡糖苷天竺葵素、3-[6-(对香豆酰)-L-葡萄苷(2→1)-葡糖苷]-5-[(6-丙二酰)-D-吡喃葡糖苷]天竺葵素。通过质谱信息和文献报道,第四种花色苷的结构应为3-[6-(阿魏酰)-L-葡糖苷(2→1)-葡糖苷]-5[-(6-丙二酰)-D-吡喃葡糖苷]天竺葵素。     

新型花色苷衍生物oxovitisin A制备条件优化及其体外抑制癌细胞增殖活性

以植物花色苷为原料,通过羧基吡喃花色苷形成及微氧化两步反应法制备出具有非氧鎓离子结构和内酯型吡喃环结构的吡喃酮型花色苷衍生物（oxovitisin A）,进行了反应温度、pH值、反应介质乙醇体积分数和反应时间影响oxovitisin A生成量的单因素试验,正交试验优化得到oxovitisin A的最佳制备条件。高效液相色谱监测反应过程,高效液相色谱-二极管阵列检测-串联离子阱多级质谱法对反应产物进行定量和定性分析。利用“罗丹明B蛋白染色法”考察了oxovitisin A以及不同结构的吡喃花色苷衍生物（羧基吡喃花色苷、甲基吡喃花色苷）、前体花色苷（锦葵花色苷）的体外抗癌细胞增殖活性。结果表明：以1.0 mg/mL的羧基吡喃花色苷为原料,oxovitisin A的最佳制备条件是pH 3.6、反应温度45 ℃、乙醇体积分数20%、反应时间21 d,各因素对oxovitisin A得率影响的大小次序为pH值＞反应温度＞反应时间＞乙醇体积分数。高效液相色谱-串联质谱结合多级质谱裂解分析表明,反应的主要产物是oxovitisin A,反应第21天得率为26.59%。抗癌细胞活性实验结果表明,oxovitisin A能显著抑制人肠癌细胞（Caco-2）和人乳腺癌细胞（MCF-7）细胞增殖,IC50分别为（238.8±24.6）、（85.7±9）μg/mL,抑制作用明显强于羧基吡喃花色苷和甲基吡喃花色苷。     

酶法提取刺五加干果花色苷工艺优化及其组成分析

目的 优化刺五加花色苷的提取条件,并对该花色苷的组成进行分析。方法 通过单因素实验研究酶的种类、液料比、酶添加量、酶解温度和酶解时间对提取液中花色苷含量的影响,利用响应面试验优化了刺五加果花色苷的提取工艺。并利用超高效液相色谱串联三重四级杆飞行时间质谱（UPLC-Triple-TOF/MS）对刺五加花色苷进行结构鉴定。结果 刺五加果花色苷提取的最佳工艺条件为：液料比18︰1 mL/g,果胶酶的添加量4.2‰,酶解温度55 ℃,酶解时间3.0 h,在此条件下刺五加果的花色苷含量达到6.00 mg/g。经过UPLC-Triple-TOF/MS分析其中主要花色苷为矢车菊素3-O-(2-O-β-D-吡喃木糖基)-β-D-吡喃葡萄糖苷。结论 利用果胶酶法来制备刺五加花色苷是可行的,所得刺五加花色苷为矢车菊素3-O-(2-O-β-D-吡喃木糖基)-β-D-吡喃葡萄糖苷。     

中国东亚种红葡萄酒的颜色特征及酚类组成研究

为探究东亚种红葡萄酒的颜色特征以及酚类物质轮廓,采用分光光度法、酶标仪和高效液相色谱-串联质谱（HPLC-MS/MS）技术分别对3款东亚种、3款欧亚种及2款杂交种（山葡萄×欧亚种）红葡萄酒的颜色和酚类物质组成进行分析,并对其花色苷衍生物进行鉴定。结果表明,与欧亚种红葡萄酒相比,东亚种红葡萄酒的辅色花色苷含量较高（55.63%～55.66%）,而聚合花色苷含量较低（19.63%～27.01%）,并且花色苷含量较为丰富（399.30～829.95 mg/L）,以花色素-3,5-O-双葡萄糖苷为主（＞97.50%）,酰化花色苷较低（＜21.78%）。花色苷衍生物检测结果表明,东亚种红葡萄酒中的花色苷与（表）儿茶素聚合产物和吡喃花色苷的含量极低（痕量水平）。东亚种红葡萄酒在非花色苷酚组成上的突出特点为单体和二聚体黄烷醇的含量极低,因此其对酒体颜色的潜在影响可能较小。     

杨梅中主要花色苷的组成与结构

采用V(甲醇)∶V(甲酸)=19∶1溶液提取杨梅中的花色苷,并用阳离子交换树脂初步纯化得到杨梅花色苷提取物,然后以水-正丁醇-甲基叔丁基醚(TBME)-乙腈-三氟乙酸(TFA)(体积比5∶2∶1.5∶1∶0.001)为溶剂系统,用高速逆流色谱对杨梅花色苷进行分离。再由C18柱色谱纯化后,得到2种主要的花色苷单体Ⅰ和Ⅱ。经ESI-MS和NMR鉴定为矢车菊色素-3-β-吡喃型葡萄糖苷(Ⅰ)和飞燕草色素-3-葡萄糖苷(Ⅱ)。另外,文中对3个杨梅品种的花色苷组成进行了分析,结果表明,矢车菊色素-3-β-吡喃型葡萄糖苷是杨梅花色苷的主要组成成分,占花色苷总量的90%以上。     

花色苷抗心血管疾病的研究进展

心血管疾病是世界范围内一个主要的死亡原因,每年对社会带来巨大的健康问题和经济负担。花色苷属于黄酮类物质,广泛存在于植物中的水溶性天然色素。花色苷具有抗氧化、抗炎、抗癌以及心血管保护等多种生物活性,本文章对花色苷的抗心血管疾病作用进行综述。     

Statistical interpretation of the color parameters of red wines in function of their phenolic composition during aging in bottle

The relationships between the color parameters (colorimetric indexes and CIELAB variables) and the phenolic components (anthocyanins, pyranoanthocyanins, hydroxybenzoic and hydroxycinnamic acids, flavanols and flavonols) of young red wines from Vitis vinifera L. cv. Tempranillo, Graciano and Cabernet Sauvignon (vintage 2000, Navarra, Spain), have been investigated during 26 months of wine aging in bottle (a period embracing their commercial life), through the application of different statistical analysis (principal component, correlation and polynomial regression). The results of the principal component analysis first indicated that for each variety a high degree of interrelation existed between the color parameters and the anthocyanin pigments. Moreover, it was found that for each variety the color parameters were correlated with the anthocyanins during aging in bottle. Finally, by the application of polynomial regression analysis, both anthocyanins (simple glucosides and acetyl-glucosides) and pyranoanthocyanins (anthocyanin-pyruvic acid adducts) were selected as the variables that best described the different color parameters during aging in bottle. However, differences were found between varieties in relation to the type of anthocyanin pigment selected for describing each color parameter, finally indicating the importance of the grape variety factor in the definition of the wine chromatic characteristics.     

Chromatic characteristics and anthocyanin profile of a micro-oxygenated red wine after oak or bottle maturation

Micro-oxygenation (MOX) was applied to a red wine for 5 months following the end of alcoholic fermentation and then both a non-oxygenated control wine and the micro-oxygenated wine were matured in oak barrels or bottles. The concentration of anthocyanin and anthocyanin-derived compounds and the chromatic characteristics of the control and micro-oxygenated wines after the maturation period were studied. Anthocyanin and anthocyanin-derived compounds composition were determined by liquid chromatography-mass spectrometry; and, together with monomeric anthocyanins, compounds including direct anthocyanin-flavanol adducts, ethyl-linked anthocyanin-flavanol compounds, and pyranoanthocyanins were identified. The results showed that the improvements in the chromatic characteristics obtained by applying the MOX technique to Monastrell wines were maintained after an aging period in bottle or barrel. The color intensity of wines increased during maturation in oak barrels, whereas the color of the bottled wines decreased, although MOX wines always showed higher color intensity than the respective control wines. Bottled wines also showed a higher increase in tint and a lower quantity of compounds resistant to SO₂ discoloration than oak matured wines, indicating that the formation of these compounds is favored by the oxidative conditions in oak barrels.     

Identification of novel pyranoanthocyanins in berry juices

In most fruit and berry products the attractive red color is unstable and easily susceptible to degradation. The colors of strawberry and raspberry juices can be enhanced and stabilized by the addition of cinnamic acids. The aim of this study was to identify the novel anthocyanin products responsible for the improved juice color. The pyranoanthocyanins formed were detected using high-performance liquid chromatography, and the fractions from this were analyzed using a nano-ESI tandem MS technique. The pyranoanthocyanins identified were formed from reactions of the main anthocyanins of strawberry and raspberry juices with ferulic acid or sinapic acid. The new anthocyanin derivatives were the 4-vinylguaiacol and 4-vinylsyringol adducts of pelargonidin and cyanidin. This is the first time that pelargonidin 3-glucoside-based vinylphenol pyranoanthocyanins and pyranoanthocyanins with more complex sugar residues have been found. This is also the first time pyranoanthocyanins have been detected in strawberry and raspberry juices.     

Electron spin resonance spectroscopy studies on the free radical scavenging activity of wine anthocyanins and pyranoanthocyanins

Anthocyanins are a group of natural occurring pigments responsible for the red-blue color of grapes and many fruits and vegetables. Anthocyanins and derived pigments are of double interest, one technological, as they can be used as natural colorants, and another one due to their implication on human health through their antioxidant activity. Although there are numerous studies regarding the antioxidant activity of grape extracts as well as red wine, the free radical scavenging activity of purified anthocyanins and pyranoanthocyanins is largely unknown. In the present study, the hydroxyl and superoxide anion scavenging activities of anthocyanins and their pyruvic acid adducts were systematically investigated by electron spin resonance spectroscopy and spin trapping. The 3-glucosides of delphinidin, cyanidin, petunidin, pelargonidin and malvidin, and the pyruvic adduct of the 3-glucoside of delphinidin exhibited a potent superoxide anion radical scavenging and, to a lesser extent hydroxyl anion radical scavenging activity. The pyranoanthocyanins of cyanidin, petunidin, malvidin and pelargonidin showed a high capacity to scavenge superoxide anion radicals but did not scavenge hydroxyl radicals. Current data indicate that formation of anthocyanin adducts with pyruvic acid, which may occur during wine ageing or fruit juice processing, decreases the hydroxyl and superoxide anion scavenging and thus could decrease the antioxidant potential of these compounds.     

Red clover isoflavonoids as anthocyanin color enhancing agents in muscadine wine and juice

Isoflavonoid extracts from red clover (Trifolium pratense) leaves were found to enhance overall color and stability of anthocyanin 3,5-diglucosides present in muscadine grape (Vitis rotundifolia) juice and wine through intermolecular copigmentation reactions. Predominant isoflavonoids present in red clover included formononetin, biochanin A, and prunetin and were the major polyphenolics identified to influence anthocyanin color and stability. Since red clover isoflavonoids have poor water solubility characteristics, this allowed for removal of extraneous non-isoflavonoid compounds using hot water and subsequent extraction with ethanol. Isoflavonoid solubility was evaluated as a function of ethanol concentration with recoveries up to 57% found in 20% solutions. Changes in maximum absorbance, total soluble phenolics, isoflavonoids, and anthocyanins were evaluated in muscadine juice and wine following the addition of isoflavonoid extracts with maximum color enhancement found at an anthocyanin to cofactor ratio of 1:8, after which their solubility was prohibitive. Additionally, dried leaves and ethanolic extracts of red clover were added prior to and following fermentation of muscadine wine (11% ethanol) stimulating the natural copigmentation that takes place during red wine fermentation and aging processes. Once fermentation was complete, finished wines were evaluated over a 9-week storage period at 20 and 37 °C. Despite low levels of isoflavonoids present, color improvement and anthocyanin stability was observed in the wines during storage. Little information is available on copigmentation reactions occurring in actual food systems, yet red clover isoflavonoids proved to be novel and effective color enhancing compounds when used in low concentrations in young muscadine wines.     

Evolution of polyphenols in red wines from Vitis vinifera L. during aging in the bottle

The evolution of anthocyanins and pyranoanthocyanins (as measured by high-performance liquid chromatography) in young red wines from Vitis vinifera L. cv Tempranillo, Graciano and Cabernet Sauvignon (vintage 2000) from Navarra (Spain) was studied during 26 months of aging in the bottle. For the anthocyanin pigments of grape origin, a progressive decrease in their concentration, corresponding to first-order kinetics, was observed during this period. Independently of the anthocyanin structure studied, grape anthocyanins in Tempranillo wine presented twofold slower kinetics than those in Graciano and Cabernet Sauvignon wines, which exhibited a very similar disappearance rate. Acylated anthocyanins presented a slightly higher disappearance rate than the nonacylated ones, indicating the possible hydrolysis of the former into the latter forms. However, no distinction was observed in the kinetics of the different anthocyanidin forms (delphinidins, petunidins, peonidins and malvidins). These results indicate that during aging under nonoxidative conditions (bottle), the chemical reactivity of grape anthocyanins in wine is influenced by the grape variety, a factor that imposes over the stability associated with the chemical structure of each anthocyanin form. In relation to the evolution of pyranoanthocyanins, anthocyanin–pyruvic acid adducts presented a similar or lower disappearance rate than their corresponding anthocyanin precursors during the first months of aging in the bottle, while anthocyanin–vinylphenol and anthocyanin–vinylflavanol adducts did not exhibit significant variations during the whole period studied.     

Anthocyanin composition in Cabernet Sauvignon red wine vinegar obtained by submerged acetification

Vinegars elaborated from white wine can be characterized by their phenolic composition. Indeed, for authenticity purpose, phenolic composition can be used to identify Sherry and Balsamic vinegars. However, the phenolic composition of red wine vinegars has scarcely been studied. Anthocyanin compounds in particular remain largely unknown. This study focuses on the analysis of anthocyanin compounds in red wine vinegar and the effect of acetification with submerged culture on such vinegars.The vinegar used in this study was produced from a young Cabernet Sauvignon wine in a laboratory-scale fermenter. Subsequent analyses of both wine and vinegar included their anthocyanin profile (by LC/MS), and their non-anthocyanin phenolic profile (by LC/DAD). In addition, wine and vinegar anthocyanin extracts were fractionated by CCC to determine the contribution of the fractions to overall antioxidant activity (AA), using ORAC, FRAP and DPPH assays.A total of 20 anthocyanin compounds were identified in the vinegar. As far as we know, this is the first time that anthocyanin-derived pigments (pyranoanthocyanins and ethyl-linked compounds) have been identified in red vinegar in such detail. Moreover, an original contribution of this study is the identification for the first time of catechyl-pyranocyanidin-3-glucoside in vinegar and wine, as well as two anthocyanin compounds not previously reported in vinegar or Cabernet Sauvignon wine: acetyl vitisin B and coumaroyl vitisin B. After the acetification process, vitisin-type and ethyl-linked compounds increased and monomeric anthocyanins, phenolic acids (ferulic acid, caffeic acid and caftaric acid) and flavan-3-ol ((+)-catechin) decreased.Although the proportion by weight of the polymeric compound fraction is similar in wine and vinegar, the AA of these polymers in vinegar is significantly greater (p < 0.05). We have also determined for the first time an approximate value of AA for malvidin-3-(6-acetyl)-glucoside isolated from vinegar.