【作者】 刘传菊；

【导师】 戚向阳；

【作者基本信息】 华中农业大学 ， 农产品加工与贮藏工程， 2008， 硕士

【摘要】 杨梅（Myrica.Rubra Sieb.et Zucc.）是我国的特产水果,杨梅中含有丰富的花色苷和黄酮类化合物等酚类物质。本文研究了杨梅中花色苷和黄酮类化合物的提取分离纯化及抗氧化特性,并对杨梅花色苷的结构、稳定性等也进行了探讨。结果表明:1.杨梅中花色苷和黄酮类化合物最佳提取条件的确定建立了杨梅中花色苷和黄酮类化合物同时提取的最佳条件,即提取温度40℃,液料比11:1,乙醇浓度70%,pH值3,在此条件下,在最佳条件下花色苷的提取率达到94.33%,黄酮类化合物的提取率达到93.72%。2.杨梅花色苷和黄酮类化合物分离纯化条件的研究探讨了三种大孔树脂（AB-8、D101和ADS-17）分离纯化杨梅花色苷和黄酮类化合物的效果,发现AB-8和D101两种大孔树脂的分离效果较好。这两种树脂分离杨梅花色苷和黄酮类化合物的最佳条件均为:将杨梅水提液浓缩后调pH值至3.0,上样后依次用水及60%乙醇（体积分数）溶液进行洗脱,收集60%乙醇洗脱液,经真空浓缩及冷冻干燥,可得到杨梅粗提物粉末,其得率为1.44%,花色苷的百分含量为22.08%,黄酮的百分含量为70.758%。葡聚糖凝胶LH-20能有效分离花色苷和黄酮类化合物,其最佳分离条件为:将大孔树脂柱分离制备的粗提物溶解上样后,先用含0.1%盐酸的20%甲醇溶液做洗脱剂,根据洗脱曲线收集洗脱液并浓缩冷冻干燥得组分1;再用60%丙酮洗脱,收集洗脱液,经浓缩干燥得组分2。组分1的得率为29.49%,花色苷的含量为73.88%;组分2的得率为46.08%,其黄酮类化合物含量为85.31%。3.杨梅花色苷的结构表征采用UV-vis,IR和HPLC-MS分析方法对纯化后的花色苷样品进行了初步结构表征。结果表明,杨梅中花色苷主要由矢车菊素-3-葡萄糖苷和芍药素-3-葡萄糖苷组成,且以矢车菊素-3-葡萄糖苷为主。4.杨梅花色苷的稳定性研究研究了杨梅花色苷在不同条件下的稳定性。结果表明:糖对杨梅花色苷稳定性的影响与糖结构和糖浓度有关。当浓度较低（＜5%）时,蔗糖、葡萄糖和果糖对杨梅花色苷均具有护色作用,其中以蔗糖的护色作用最佳;当浓度逐渐增加时,蔗糖和葡萄糖的护色作用趋于稳定;而果糖却促进了杨梅花色苷的降解,当浓度达到20%时,杨梅花色苷的保存率仅为53.49%。茶多酚和芦丁对花色苷有一定的护色和辅助成色作用。花色苷在pH值较低即pH≤4时比较稳定,当pH＞4时,花色苷破坏严重。随温度升高,杨梅花色苷溶液保存率逐渐降低,花色苷的降解加剧;100℃下杨梅中花色苷的热降解遵循动力学一级反应,并且其半衰期t_1/2=133.8247 min,降解速率常数k=0.005262;受热时间对杨梅花色苷溶液的Hunter a值有明显影响,受热时间越长,杨梅花色苷溶液的Hunter a值越低,杨梅花色苷的降解加剧。Fe³⁺、Fe²⁺和Al³⁺可与杨梅中的花色苷络合引起变色,而Ca^2a、CH²⁺、Na⁺、K⁺对杨梅中的花色苷的影响不明显。杨梅花色苷在有光的条件下不太稳定。过氧化氢和抗坏血酸能显著促进杨梅花色苷的降解。牛血清白蛋白能与花色苷形成聚合物,阻止花色苷的降解,起到一定的护色作用。在杨梅酒发酵过程中加入300 mg/L的芦丁或茶多酚均对杨梅酒起到一定的护色作用,且芦丁的护色效果又稍微优于茶多酚。因此,在实际生产时,可在杨梅酒发酵前加入少量芦丁来保护杨梅酒的色泽。5.杨梅酚类物质的抗氧化活性采用不同体系研究了不同杨梅提取物的抗氧化作用,发现样品1（酚类粗提物）、样品2（花色苷部分）、样品3（黄酮部分）、样品4（杨梅汁粉末）是有效的抗氧化剂,在所测的四个体系中,均显示出较高的抗氧化活性。其中样品1的抗氧化活性最高,在清除超氧阴离子、羟基自由基和DPPH自由基的作用上均高于抗氧化剂标准对照品EGCG和Vc,其对过氧化氢的清除能力上,虽略低于EGCG,却高于Vc。样品2（花色苷）对DPPH的清除作用高于样品3（黄酮类化合物）,而在对超氧阴离子、羟基自由基和H₂O₂的清除活性上低于样品3,表明杨梅中的黄酮和花色苷均有很好的抗氧化活性,且两者的抗氧化作用具有一定的协同增效作用。更多还原

【Abstract】 Chinese bayberry（Myrica.Rubra Sieb.et Zucc.） is a specific fruit species in China.There are plenty of anthocyanins and flavonoids in it.In this paper,the extraction,isolation,purification and antioxidant activity of anthocyanins and flavonoids in bayberry were studied,and the structure and stability of anthocyanins were also researched.The results were as follows:1.Determination of the optimal extraction condition of anthocyanins and flavonoids in bayberryThe optimal extraction condition of anthocyanins and flavonoids in bayberry was established.When the material was extracted with 70%ethanol（pH3） at 40℃and at the ratio of liquid to material（v/w） 11:1,the extraction rate of anthocyanins and flavonoids was 94.33%and 93.72%respectively.2.Isolation and purification of anthocyanins and flavonoids from bayberryThe separation effects of three macro-porous resins（AB-8,D101 and ADS-17） on anthocyanins and flavonoids from bayberry were studied.It is found that the separation effects of AB-8 and D101 were better.The optimal conditions of these two resins both were:the extraction concentration of bayberry was adjusted to pH 3.0 and applied to AB-8 and D101 macro-porous resin,eluted with distilled water and 60% ethanol at the natural flow rate and 60%ethanol elution was collected,concentrated and freeze-dried to give extracted powder.The product yield was 1.44%and the contents of anthocyanins and flavonoids were 22.08%and 70.758%respectively.Sephadex LH-20 was used to further separate anthocyanins and flavonoids from bayberry extract.The bayberry extract with resins was applied to a Sephadex LH-20 column.The column was eluted with 20%methanol（contain 0.1%HCl） and 60% acetone solution successively,the main chromatography peaks with 20%methanol as eluting solution was collected to give product 1,and 60%acetone eluting solution was collected to give product 2.The yield of product 1 was 29.49%,its anthocyanins content was 73.88%;the yield of product 2 was 46.08%,its flavonoids content was 85.31%.3.Structure identification of anthocyanins from bayberryThe anthocyanins in product 1 were identified by the UV-vis,IR,and HPLC-MS. The results showed that cyanidin-3-glucoside and peonidin-3-glucoside were the main anthocyanins of bayberry,and the cyanidin-3-glucoside content was more.4.The stability of anthocyanins in bayberryThe stability of bayberry anthocyanins under different conditions were studied. The effect of sugar on the stability of anthocyanins was related to the structure and concentration of sugar.When the sugar concentration was low（＜5%）,sucrose, glucose and fructose all could protect the color of anthocyanins from bayberry,and sucrose is best.With increase the concentration,the protective effects of sucrose and glucose on anthocyanins were stabilities whereas fructose accelerated the degradation of anthocyanins,the preservative rate of anthocyanins was merely 53.49%when the fructose concentration was 20%.Tea polyphenols and rutin could protect color and have a adjuvant function.Anthocyanins were stable at low pH（pH≤4）,but degradated strongly when pH＞4.The preservative rate of anthocyanins decreased gradually with the increase of temperature,the thermal degradation of anthocyanins according to the first order kinetic reaction with half-life(t_1/2) 133.8247min and degradation constant（k） 0.005262.The heating time has an obvious effect on the Hunter a value of the anthocyannins solution,the hunter a value was decreased with the heating time increase.Fe³⁺、Fe²⁺ and Al³⁺ could complex with anthocyannins and change color of anthocyanins,but Ca²⁺、Cu²⁺、Na⁺、K⁺ did not had any effects on anthocyannins color.Anthocyanins were sensible to light,and hydrogen peroxide and ascorbic acid could accelerate the degradation of anthocyanins remarkably.Bovine serum albumin（BSA） could complex with anthocyanins to form polymer and had some protection effect.In the fermentation process of bayberry wine,300 mg/L rutin or tea polyphenols had some protection effect on wine color.5.Antioxidant effects of polyphenols extract from bayberryDifferent systems were used to study the anti-oxidative activity of different bayberry’s extracts.The results showed that sample 1（phenolic extract）,sample 2 （anthocyanins fraction）,sample 3（flavonoids fraction） and sample 4（the powder of bayberry juice） were all effective antioxidants.They all showed high anti-oxidative activities in these four systems.Sample 1 had the best anti-oxidative activity,its scavenging activities on superoxide anion,hydroxyl radical and DPPH were higher than that of EGCG and Vc,but its scavenging effect on H₂O₂ was lower than EGCG. The scavenging activity on DPPH of sample 2（anthocyanins fraction ） was stronger than sample 3（flavonoids fraction）,but its scavenging activities on superoxide anion, hydroxyl radical and H₂O₂ were lower than that of sample 3（flavonoids fraction）.The results hinted that anthocyanins and flavonoids from bayberry both had high anti-oxidative activities,and these two compounds also had synergistic effect.更多还原