【作者】 王二雷；

【导师】 殷涌光；

【作者基本信息】 吉林大学 ， 农业机械化工程， 2014， 博士

【摘要】 蓝莓产业在国际及国内市场上已经展现出巨大潜力，尽管我国的蓝莓产业起步较晚，但最近几年已经成为我国发展最快的新型产业之一。近些年，国内外针对蓝莓中花青素的研究大部分局限于花青素的结构鉴定及粗提物的制备研究工作上，对制备高纯度花青素及单体的研究并不多见。在花青素的抗肿瘤活性研究方面，国外已有研究表明花青素具有抑制肿瘤增殖和阻断致癌因子等作用，但对花青素及苷元在抑制黑色素瘤方面的研究报道还为数不多。本文开展了以下几个方面的研究：（1）优化了蓝莓花青素的含量检测方法和结构鉴定方法；（2）建立了纯天然的制备高纯度蓝莓花青素提取物的方法；（3）研究了蓝莓花青素单体物质的纯化方法；（4）利用化学降解途径制备出蓝莓花青素苷元，并进行结构鉴定及稳定性评价；（5）利用体外、体内活性实验初步研究蓝莓花青素的抗肿瘤作用。首先利用pH示差法及高效液相色谱法对蓝莓果实中花青素进行了含量分析。利用pH示差法测得长白山野生蓝莓果实中含花青素144.72±4.49mg/100g鲜果，栽培种蓝莓中含216.50±6.26mg/100g；利用HPLC法测得野生蓝莓中花青素含量为173.15±8.16mg/100g鲜果，而栽培种中含245.52±11.54mg/100g鲜果。通过比较，发现两种方法均适用于花青素的含量分析，前者偏重于总花青素含量测定，后者更适合单体花青素的含量分析。在测得花青素总含量的基础上，利用UV-vis、HPLC及HPLC-DAD-ESI-MS三种技术对我国野生及栽培种蓝莓中花青素进行了种类鉴定及含量分析。从野生蓝莓果实中鉴定出13糖苷化的花青素，从栽培种中鉴定出12种糖苷化的花青素及2种酰化的花青素。在野生蓝莓果实中锦葵色素-3-O-葡萄糖苷含量最丰富，约占总花青素的28%，含量为48.44mg/100g鲜果；栽培种中含量最丰富的为锦葵色素-3-O-半乳糖苷，约占总花青素21%，含量为51.45mg/100鲜果。研究了蓝莓花青素的提取、萃取、大孔树脂纯化、凝胶色谱纯化工艺。在提取工艺中，发现70%的盐酸化乙醇适用于野生蓝莓中花青素的提取工艺，但不适合于栽培种中含酰化形式的花青素的提取。在花青素的萃取工艺中，经过3次萃取后，乙酸乙脂能有效去除花青素粗提液中大部分的黄酮类物质。在Amberlite XAD-7HP大孔树脂纯化蓝莓花青素的过程中，利用35%的酸化乙醇洗脱层析柱后，花青素的纯度达32%，且从中未检出非花青素的黄酮类物质。在蓝莓花青素粗提物经过Sephadex LH-20凝胶层析后，发现25%的酸化乙醇能将粗提物分离成3段稳定的花青素组分，且每段组分中花青素纯度分别为59.53%，68.04%和65.79%；利用高效液相色谱对这3段组分进行鉴定后，得出组分1中含量最丰富的物质为锦葵色素-3-O-葡萄糖苷，占总峰面积的86.8%；组分2中含量最丰富的为矮牵牛色素-3-O-葡萄糖苷（47.3%）；组分3以飞燕草素-3-O-葡萄糖苷单体为主，占总峰面积的51.6%。利用半制备型C18小柱（4.6×250，5μm）对Amberlite XAD-7纯化出的蓝莓花青素粗提物的色谱条件进行了初步筛选。在此基础上开展了中型半制备色谱柱(20.0×250mm,5μm)的色谱条件优选，采用经LH-20色谱柱纯化出的3种组分进行了半制备液相色谱柱条件优化，通过二次进样方法制备出3种高纯度的蓝莓花青素单体。利用UV-vis、HPLC及HPLC-ESI-MS对三种花青素单体进行了结构鉴定及纯度测定，得出锦葵色素-3-O-葡萄糖苷的纯度为97.7%，矮牵牛色素-3-O-葡萄糖苷的纯度为99.3%，飞燕草素-3-O-葡萄糖苷的纯度为95.4%。制备出的三种花青素单体均可以作为花青素的对照品用于其它天然产物中的花青素成分分析，也能够为生产蓝莓高附加产品提供必要的原材料。此外，本实验优化出的半制备液相方法简单，为扩大规模生产花青素的标准品提供良好的参数基础。运用UV-vis法比较了蓝莓花青素糖苷化形式与降解后的苷元形式在结构上的区别，得出苷元形式花青素在可见光区的最大吸收波长为521nm左右，比糖苷形式的花青素蓝移14nm左右。当盐酸浓度在2~4mol/L之间、水解时间在40~60min时，可完全水解花青素，得到苷元形式的花青素。利用高效液相色谱法测得野生蓝莓和栽培种蓝莓果实花青素粗提液水解后均得到5种基本的花青素苷元，且均以飞燕草素衍生物为主，矢车菊素衍生物为辅。为研究蓝莓花青素的抗肿瘤效果，我们首先比较了蓝莓花青素样品的抗氧化作用。我们考察了花青素对DPPH和ABTS自由基清除能力，得出蓝莓花青素粗品具有较强的抗氧化作用，组分1、Vc的抗氧化能力均较低。利用蓝莓花青素粗品研究了花青素的抗肿瘤作用。在外体实验中，得出蓝莓花青素浓度在200~1000μg/mL范围内时，对黑色素瘤细胞有较强的抑制作用；在体内实验中，首先建立了小鼠黑色素瘤模型，得出2000μg/mL的蓝莓花青素样品对小鼠肺部黑色素瘤的抑制作用高于20μg/mL的DOX（阿霉素）阳性对照品，而200μg/mL的蓝莓花青素样品与DOX的作用差别不大。通过对不同组小鼠的肺部进行重量大小比较，得出黑色素瘤对脏器的侵袭会造成其重量的增加，但这种侵袭不会对小鼠的总重造成影响。本文系统地分析了蓝莓中花青素含量及种类，构建了纯天然的花青素分离纯化工艺，对蓝莓花青素的抗肿瘤作用进行了体外和体内活性评价。本实验的开展有助于揭示蓝莓花青素潜在的营养价值，能够为深入研究蓝莓花青素的结构及功能活性奠定基础，预期为我国蓝莓产业高附加值产品的开发提供一条新的途径，为拓展抗肿瘤作用的保健食品或纯天然药品的开发提供理论依据。更多还原

【Abstract】 Nowadays, Blueberry industry is showing great potential in the international anddomestic market. Despite the late start of the blueberry industry in our country, it has beenone of the fastest growing new industries. In recent years, most of the researches onanthocyanins from blueberries limited to structural identification and preparation ofanthocyanin crude extracts, there are only a few studies on preparation of high purity ofanthocyanin extracts and monomers. On the aspect of anti-tumor activity of anthocyanins,abroad studies have showed that anthocyanins could inhibit tumor growth and blockcarcinogens, however, the effects of anthocyanins and acylcones on the inhibition ofmelanoma cell research have been rarely reported. This paper focuses on the followingresearch aspects:(1) optimized the content and structure detection methods of anthocyanins,(2) established a natural process for producing high-purity blueberry anthocyanin extract,(3)studied the purification of blueberry anthocyanins and monomers,(4) prepared anthocyaninacylcones using chemical degradation pathway, and then evaluated the stability and chemicalstructure,(5) explored the in vitro and in vivo anti-tumor effects of blueberry anthocyanins.Firstly, both pH differential method and HPLC method employed to determine the totalanthocyanins content (TAC) of blueberry fruits. Through the pH differential method, theTAC in Wild blueberries blueberry fruit reached up to144.72±4.49mg/100g fresh fruit, andthe TAC was216.50±6.26mg/100g in cultivated blueberry fruits. Through the HPLCmethod, the TAC in wild blueberry was173.15±8.16mg/100g fresh fruit, compared with245.52±11.54mg/100g fresh fruit in cultivated blueberry. By contrast, both methods areapplicable to the content analysis of anthocyanin, what is more, the former is more feasiblefor the determination of total content, and the latter is more suitable for the analysis ofmonomeric anthocyanins.Based on the understanding of total anthocyanin content, three techniques of UV-vis,HPLC and HPLC-ESI-MS methods were combined to analyze the anthocyanin profiles inwild and cultivated blueberries. There were13glycosylated anthocyanins in wild blueberry,and12glycosylated and two acylated anthocyanins in cultivated blueberry species.Malvidin-3-O-glucoside is the most abundant in the wild blueberry, accounting for28%of the total anthocyanin content, reaching up to48.44mg/100g fresh fruit, while malvidin-3-O-galactoside is the predorminent monomer, accounting for21%of the total anthocyanincontent, reaching to51.45mg/100fruit.The separation process of blueberry anthocyanins was carried out using extraction,partition, macroporous resin purification, gel chromatography purification. In the extractionprocess, it was found that70%ethanol acidified with hydrochloride was suitableanthocyanins extraction, which is not suitable for the extraction of acylated anthocyaninsfrom cultivated blueberries. In the partition process, ethyl acetate could effectively removemost of the non-anthocyanin flavonoids from the crude extracts of anthocyanin after threetimes of partition. In the process of Amberlite XAD-7HP macroporous resin purification,35%ethanol was used to elute the anthocyanins, and the purity of free-dried anthocyaninsapproached32%where the non-anthocyanin flavonoids were not detected. In the process ofSephadex LH-20gel column chromatography, it was found that the crude extracts ofanthocyanins could be successfully separated into three segments using25%ethanol as theeluent, their purities were59.53%,68.04%and65.79%, respectively. Through an analysis ofHPLC-PDA, segment one mainly contained malvidin-3-O-glucoside (86.8%), segment twocontained petunidin-3-O-glucoside (47.3%), segment three contained delphinidin-3-O-glucoside (51.6%).A small semi-preparative C18column (4.6×250,5μm) was tested to purify the crudeextracts of blueberry anthocyanins. And then, the medium-sized semi-preparative C18column (20.0mm×250mm,5μm) was used to isolate the components obtained fromSephadex LH-20. Three high-purity blueberry anthocyanin monomers were prepared aftertwice injection. The profiles and purities of three anthocyanin monomers were determinedusing combined techniques, such as UV-vis, HPLC and HPLC-ESI-MS. Three monomerswere malvidin-3-O-glucoside (purity:97.7%), petunidin-3-O-glucoside (purity:99.3%) anddelphinidin-3-O-glucoside (purity:95.4%), respectively. All of the three monomers can beused as references for anthocyanin analysis from other natural products, even the productionof high added blueberry products. Furthermore, the semi-preparative HPLC process in thispaper can lay a foundation for the scale production of anthocyanin standards.The UV-vis spectrums between the blueberry anthocyanin glycosides and aglycone forms were studied.The maximum absorption wavelength of anthocyanin aglycone was521nm, which blueshifted14nm compared with anthocyanins glycosides. When theconcentration of hydrochloric acid was2to4mol/L, hydrolysis time was40to60min, it cancompletely hydrolyzed anthocyanins glycosides. There were five basic anthocyaninaglycones in wild and cultivated blueberries, and delphinidin derivatives are the mostaffluent, followed by cyanidin derivatives.In order to evaluate the anti-tumor effects of blueberry anthocyanins, we first comparedthe antioxidant capacities of blueberry anthocyanin samples. We investigated the DPPH andABTS radical scavenging abilities, and found that the crude extracts of blueberryanthocyanins have stronger antioxidant ability than that of other materials, and the segmentone and Vitamin C ranked the last. Further, the crude extracts were used to evaluate theanti-tumor effect of blueberry anthocyanins. In vitro experiments, the concentration ofanthocyanins in the range of200~1000μg/mL could strongly inhibited the proliferation ofmurine melanoma cells. in vivo experiments, the murine melanoma model was firstestablished, and the results showed that2000μg/mL of anthocyanin extracts showed a higherinhibition capacity on murine melanoma cells than20μg/mL of DOX (doxorubicin) group,while200μg/mL of anthocyanin extracts were not better than DOX group. Through acomparison on the lung weight among different groups, the invasion of melanoma couldcause an increase in organs weight, but which could not affect the total weight of mice.This paper systematically analyzes the contents and profiles of anthocyanins inblueberries. We established a natural process for separation and purification of blueberryanthocyanins and then explored the anti-tumor effects of blueberry anthocyanins in vitro andin vivo. The present results will help to reveal the potential nutritional value of blueberries,and lay a foundation for the further study on the structure and functions of blueberryanthocyanins. It is expected to provide a new way for the development of high value-addedproducts of blueberry anthocyanins, to expand the application of anti-tumor foods or naturalmedicines.更多还原