【作者】 方芳；

【导师】 蒋益虹；

【作者基本信息】 浙江大学 ， 食品科学， 2014， 硕士

【摘要】 作为一种蔬菜粮食兼用作物,马铃薯因含有丰富的淀粉、多种生物活性成分、良好的适口性和加工性广受人们喜爱。紫色马铃薯作为一种特色马铃薯,含有丰富的花色苷,可作为天然食用色素的来源。据报道紫色马铃薯具有清除自由基、预防肿瘤、糖尿病等生物功能,因而逐渐引起人们的关注。在西方发达国家,前列腺癌是发病率和致死率仅次于肺癌的第二大癌症,在我国患病人数呈现逐年递增的趋势。前列腺癌后期通常会由激素依赖性转变激素非依赖性前列腺癌,后者对化疗不敏感,是导致绝大多数患者死亡的原因。本文以甬紫一号紫色马铃薯(Solanum Tuberosum L.)不同部位——皮和肉的花色苷提取物为研究对象,研究其抗氧化成分含量与抗氧化活性强弱,并作用于雄激素非依赖性前列腺癌Du145、PC-3细胞,研究其对两种癌细胞的抑制增殖、诱导凋亡作用机制,并进一步探索紫色马铃薯皮花色苷提取物发挥抗前列腺癌功效的关键有效成分。研究结果为紫色马铃薯抗癌生物功能的开发奠定了理论基础,并对花色苷体外抗前列腺癌的研究有一定的借鉴意义。主要研究结论如下：(1)以紫色马铃薯皮和肉为研究对象,通过提取、纯化分别获得了花色苷提取物(Anthocyanin Extracts from Purple Potato Peel, AEPPP;Anthocyanin Extracts from Purple Potato Flesh, AEPPF)。测定了紫色马铃薯皮和肉花色苷提取物中主要的抗氧化成分含量,分别为：总酚606.50±31.74、593.86±21.91mg/g冻干粉(以绿原酸计),总黄酮472.97±2.26、353.02±2.64mg/g冻干粉(以芦丁计),花色苷207.33±6.67、320.81±4.32mg/g冻干粉(以矢车菊素-3-葡萄糖苷计)。实验表明,紫色马铃薯皮和肉的花色苷提取物均具有一定的抗氧化活性。AEPPP与AEPPF清除自由基DPPH、 ABTS+能力与铁离子还原能力均弱于对照Trolox, AEPPP与AEPPF的抗氧化活性差异不明显。AEPPP与AEPPF清除DPPH自由基的IC50值分别为207.41μg/mL、241.61μg/mL,清除ABTS+自由基的IC50分别为96.99μg/mL、109.65μg/mL,铁离子还原能力分别是对照Trolox的0.54倍和0.45倍。。(2)紫色马铃薯皮花色苷提取物能有效抑制雄激素非依赖性前列腺癌Du145、PC-3细胞的增殖,并呈明显的剂量-时间依赖效应,作用于Du145细胞24、48、72h的IC50值分别为39.02μg/mL、33.81μg/mL、26.71μg/mL,作用于PC-3细胞24、48、72h的IC50值依次为50.03μg/mL、42.95μg/mL、33.58μg/mL,但紫色马铃薯肉的花色苷提取物效果不明显。AEPPP能够显著影响Du145、PC-3细胞的周期分布,分别将其阻滞于G0/G1期、G2/M期,从而抑制细胞增殖。PI/Annexin-VFITC双染结果显示AEPPP可诱导Du145、PC-3细胞凋亡,免疫印迹实验进一步表明AEPPP作用48h下的Du145细胞内procaspase-3活化,其表达随AEPPP浓度增加而增强,且有剪切的aivtive caspase-3出现,而PC-3细胞内未出现procaspase-3的活化,且未检测到active caspase-3。推测AEPPP诱导Du145、PC-3细胞凋亡分别通过caspase依赖途径和caspase非依赖途径。(3)探索了紫色马铃薯皮花色苷抗前列腺癌的构效关系。采用HPLC-DAD-ESI-MS/MS方法,对紫色马铃薯皮花色苷组成进行了快速鉴定,共检出14种花色苷,所含花色苷均是以五种花色素苷元——矮牵牛素、芍药素、锦葵素、飞燕草素、矢车菊素的不同形式(糖基取代/糖基取代且酰基取代)存在。有4种花色苷未进行酰基取代,形式为花色素-3-O-芸香糖苷-5-O-葡萄糖苷,含量甚微；占据较大比例(79.5%)的存在形式为花色素-3-O-对香豆酰芸香糖苷-5-O-葡萄糖苷,也存在4种花色苷其参与酰化的有机酸替换为咖啡酸或阿魏酸；此外,存在一种对香豆酸酰化的花色苷,其糖基取代形式并非常规的C3,5-位取代,而是C3,7-位取代。通过半制备液相获得三种含量较高的花色苷单体,分别为矮牵牛素-3-O-对香豆酰芸香糖苷-5-O-葡萄糖苷(petanin)、矮牵牛素-3-O-阿魏酰芸香糖苷-5-O-葡萄糖苷(petanin/f,简称)、芍药素-3-O-对香豆酰芸香糖苷-5-0-葡萄糖苷(peonanin),高效液相图谱面积归一化分析三者纯度依次为92.97%、73.60%、86.72%。MTT实验结果表明,三种花色苷单体无论单独添加还是复配添加,对Du145细胞均没有明显的增殖抑制作用,推测紫色马铃薯皮花色苷提取物中发挥关键作用的成分并非这三种含量较高的花色苷。(4)采用LH-20凝胶层析柱对紫色马铃薯皮花色苷提取物进行分离纯化,进一步探索提取物中发挥抗前列腺癌Du145细胞的关键组分。确定了层析条件为以0.1%HC1的30%甲醇溶液为洗脱剂,上样量30mg,流速1.0mL/min。层析分离后,获得六个组分AEPPP1-AEPPP6,经MTT实验分析,六个组分对Du145细胞均没有明显的增殖抑制作用,抑制效果远弱于AEPPP.但将六个组分重新混合后抑制效果显著提高,作用于Du145细胞的IC50值为175.87μg/mL,说明紫色马铃薯皮花色苷提取物中的各种有效成分相互协同实现显著的抑制Du145细胞增殖的效果。更多还原

【Abstract】 As a crop for food and vegetables, potatoes are popular because of being a rich source of starch and containing good quantity of bioactive compounds, good palatability and machinability. Purple potatoes are new developed variaities called specialty potatoes, which are rich in anthocyanins and regarded as a good source of natural food colorant. A great deal of attention has been paid to purple potatoes. It’s reported that purple potatoes could scavenge free radicals, prevent tumors and diabetes, et al. In western developed countries, prostate cancer is the second leading cancer of which morbidity and mortality rank after lung cancer. In China, the incidence of prostate cancer has presented an increasing trendency. The advanced prostate cancer usually transforms from the hormone dependent tumor into the hormone independent tumor. The latter is non-sensitive to chemical therapy, which causes the death of most patients. In this study, the anthocyanin extracts from different parts-peel and flesh of Yong Zi I purple potato (Solanum Tuberosum L.) have been the research object, of which the antioxidant contents and antioxidant activity have been investigated. The antiproliferation properties against androgen independent prostate cancer cell lines Du145and PC-3, and effects on the cell cycle distribution, the mechanism of inducing apoptosis of the anthocyanin extracts from purple potatoes have been emphatically studied. The anti-prostate cancer key components have also been further determined. This study can provide theory basis for the development of purple potato biological function against tumor. What’s more, this would have certain reference for the study of anthocyanin against prostat cancer in vitro. The results were as follows:(1) The anthocyanin extracts from purple potato peel and flesh (AEPPP and AEPPF) were extracted and purified. Then the main antioxidant contents were measured, which contained total phenolics606.50±31.74and593.86±21.91mg/g lyophilized powder (calculated as chlorogenic acid), total flavones472.97±2.26and353.02±2.64mg/g lyophilized powder (calculated as rutin), and anthocyanins207.33±6.67and320.81±4.32mg/g lyophilized powder (calculated as cyanidin3-O-glucoside) in AEPPP and AEPPF respectively. As the results suggested, anthocyanin extracts from peel and flesh of purple potato had certain antioxidant capacity. There was insignificant difference between AEPPP and AEPPF of the scavenging effects on DPPH and ABTS+radical, and ferric ion reducing power. However, they were both less effective compared with Trolox. The IC50values of scavenging DPPH radical of AEPPP and AEPPF were207.41μg/mL,241.61μg/mL, and for ABTS+radical were96.99μg/mL and109.65μg/mL. The ferric ion reducing power of AEPPP and AEPPF were0.54and0.45times of Trolox respectively.(2) The anthocyanin extracts from purple potato peel (AEPPP) inhibited the proliferation of androgen independent prostate cancer cells Du145and PC-3in a dose and time dependent manner. The IC50values of Du145cells treated with AEPPP for24,48,72hours were39.02μg/mL,33.81μg/mL and26.71μg/mL respectively. For PC-3cells, the IC50values were50.03μg/mL,42.95μg/mL and33.58μg/mL. However, the anthocyanin extracts from purple potato flesh (AEPPF) had no obvious inhibiting effects. AEPPP could significantly change cell distribution of Du145and PC-3cells. Results showed cell cycles of Du145and PC-3were arrested in G0/G1phase and G2/M phase respectively. PI/Annexin-V FITC staining indicated that AEPPP could induce apoptosis of Du145and PC-3cells. Western blotting further suggested that the expression of procaspase-3in Du145cells was strengthened and active caspase-3was detected. While in PC-3cells, there were no significant viaration of procaspase-3expression and no ocurrance of active caspase-3. The cytotoxic avtiviteis of AEPPP in Du145and PC-3cells were presumably due to activation of caspase-dependent apoptosis and caspase-independent apoptosis.(3)The structure-function relationship of anthocyanins extracted from the peel of purple potato was explored. The anthocyanin composition was rapidly characterized by HPLC-DAD-ESI-MS/MS. Fourteen kinds of anthocyanins were detected, with different substituents (glycosyl/glycosyl with acyl) of petunidin, peonidin, malvidin, delphinidin and cyanidin. Among all the anthocyanins, four kinds existed in the form of aglycon-3-rutinoside-5-O-glucoside, and nine in the form of aglycon-3-coumaroyl (/caffeoyl/feruloyl) rutinoside-5-O-glucoside. Besides, an anthocyanin was inferred with glycosyl substitutions at C3, C7site. By Pre-HPLC, three kinds of higher content anthocyanin monomers were obtained, which were3-O-p-coumaroylrutinoside-5-O-glucosides of petunidin (petanin),3-O-p-feruloylrutinoside-5-O-glucosides of petunidin (named as petanin/f), and3-O-p-coumaroylrutinoside-5-O-glucosides of peonidin (peonanin). By HPLC area normalization method, the purity of three anthocyanins was determined, whcih was92.97%,73.60%and86.72% respectively. MTT asssy suggested that neither individual use nor conpound use of three anthocyanins could suppress the growth of Du145cells. Thus, it’s speculated that the key components in AEPPP might not be the three maximum content anthocyanins.(4)To determine the key component, Sephadex LH-20was applyed to purify AEPPP. Chromatographic conditions were optimized with eluant as30%methonal containing0.1%HC1, sample amount30mg and elution velocity1.0mL/min. Six parts AEPPP1-AEPPP6were colleted through chromatographic seperation. MTT assay indicated that none of six parts had the same proliferation suppression effect as AEPPP. However, when six parts mixed, good inhibition ability was measured, of IC50value as175.87μg/mL. A variaty of bioactive compounds may inhibit the proliferation of prostate cancer Du145cells by synergy.更多还原