【作者】 田洪磊；

【导师】 张灏；

【作者基本信息】 江南大学 ， 食品科学， 2013， 博士

【摘要】 小白杏杏仁油是新疆少数民族居民长期食用的特色药食两用资源，新疆维吾尔民族居民长期将其作为一种维吾尔药物进行补脑安神、抗炎症、抗突变及抑菌等方面的传统应用，目前小白杏种植结构老化、野生品种濒危及精深加工与副产物综合利用程度低等问题已成为制约新疆小白杏杏仁油产业发展的瓶颈。在区域化特色资源的生物学功能受到普遍关注的今天，关于新疆药食两用资源的小白杏杏仁油指纹图谱及抗氧化、免疫调节作用等方面的研究尚属空白。本论文在对新疆小白杏种植资源调查统计的基础上主要开展了如下内容研究：首先，采用前期研究成果对新疆不同产地的小白杏杏仁油进行分离提取，结合GC-MS技术对制备样品进行主要生物活性物质组成分析，确认其指纹图谱中的特征指纹信息，进而采用系统聚类和相似度分析法构建生物活性物质指纹图谱库，同时结合主成分(PCA)和偏最小二乘判别分析(PLS-DA)的鉴别模型对小白杏杏仁油种类和品质进行鉴别和监控；其次，以库车小白杏杏仁资源为研究样本，实施不同方法小白杏杏仁油制备样品的生物活性物质色谱分析，考察新疆不同小白杏杏仁油样品对DPPH自由基、羟基自由基、超氧阴离子自由基及ABTS自由基等氧化应激生物标志物的清除效果，结合PLSR分析法对复杂生物活性物质组成的小白杏杏仁油进行抗氧化活性功效关系研究；再次，建立免疫力低下试验大鼠模型，以抗氧化性能研究过程中建立的超临界CO2萃取制备样品为基料，对照空白喂养组(BS)、模型组(MC)、模型小白杏杏仁油样品组(SF)实施小白杏杏仁油免疫调节作用研究，通过免疫脏器指数、血清(肝脏)免疫因子指标及免疫组织器官病检考察新疆小白杏杏仁油对免疫力低下大鼠模型的免疫调节作用；最后，以小白杏杏仁油免疫调节作用研究过程中平行获得的各对照组试验大鼠血清样本为研究对象，采用iTRAQ标记及高效液相色谱-电喷雾串联质谱(LC-ESI-MS/MS)技术对试验大鼠血清差异表达蛋白进行定量鉴定，并通过表达模式聚类(EPC)及代谢通路(Pathway)等分析对鉴定获得的差异蛋白进行功能注释，确定其参与的最主要生化代谢途径和信号转导途径。研究获得的具体成果或结论如下：（1）采用GC-MS与聚类分析相结合的方法从15个小白杏杏仁油基础样本中筛选出13个模式样本，建立了能够反映新疆小白杏杏仁油特征成分的指纹图谱，结合随机选择的10种掺假小白杏杏仁油样本及其它油脂产品进行指纹图谱相似度和PCA分析，充分论证了所建指纹图谱应用于小白杏杏仁油样品(产品)品质评价的有效性；采用PLS-DA法分别建立的不同种类油脂和不同品种杏仁油定性判别模型的预测值与实测值相关系数均在0.95以上，对检测集样本的识别率为100%，表明小白杏杏仁油指纹信息与PLS-DA判别分析相结合的方法亦可实现小白杏杏仁油和其它油脂的有效检测及准确判别。（2）通过GC及GC-MS技术对超临界CO2萃取(SCDE)、机械压榨(MCE)、超声波辅助提取(UAE)及索氏提取(SE)四种库车小白杏杏仁油制备样品进行了分析，共筛选获得了脂肪酸组分(19种)、甾醇及萜烯类(14种)等33种均呈显著差异性(P﹤0.05，α-生育酚除外)的共有活性物质；不同方法制备样品对DPPH、羟基、超氧阴离子及ABTS4种自由基均存在差异的清除能力，SCDE及MCE制备样品对相关自由基的清除能力有超越VE对照组的趋势，尤其对超氧阴离子自由基的清除效果最为显著。（3）采用PLS2模型分析了不同制备样品与抗氧化指标之间的相关性。UAE制备样品对4种自由基的清除能力相对较差，SCDE样品与氧化应激指标DPPH自由基及羟基自由基具较强的正相关性；与SCDE和MCE小白杏杏仁油制备样品相比，SE制备样品与4种抗氧化指标亦具有正相关性，但其相关性相对较弱。（4）采用PLS1模型分析了具体活性物质与抗氧化指标之间的相关性。DPPH自由基清除能力与顺式-油酸△9、亚油酸△9,12、亚油酸△8,11、亚麻酸△6,9,12、花生三烯酸△8,11,14、α(γ)-生育酚、菠菜甾醇△7,22、β-谷甾醇△5和角鲨烯△2,6,10,14,18,22呈显著正相关；羟基自由基的清除能力与亚油酸△9,12、△5-豆甾醇△5,22、β-谷甾醇△5、γ-生育酚和角鲨烯△2,6,10,14,18,22呈显著正相关；超氧阴离子自由基清除能力与棕榈油酸、花生四烯酸△5,8,11,14、α-生育酚、△5-燕麦甾醇△5,24(28)、△7-燕麦甾醇△7,24(28)呈显著正相关；ABTS自由基的清除力与反式-油酸△9、亚油酸△9,12、亚麻酸△6,9,12、α-生育酚、△5-燕麦甾醇△5,24(28)和角鲨烯△2,6,10,14,18,22呈显著正相关。角鲨烯△2,6,10,14,18,22与亚油酸△9,12及α-生育酚分别与3种以上抗氧化指标存在明显相关性，可以推测其将成为抗氧化复合产品的重要基础物质，显著正相关性活性物质中角鲨烯△2,6,10,14,18,22、花生四烯酸△5,8,11,14、△5-燕麦甾醇△5,24(28)、亚麻酸△6,9,12可能分别在不同小白杏杏仁油自由基清除体系中起关键协同作用。（5）通过免疫力低下试验大鼠模型构建及酶联免疫吸附检测的方法发现，新疆小白杏杏仁油可显著提高免疫力低下大鼠模型(MC)的脾脏指数、胸腺指数及肝脏指数(P＜0.05)，改善相关免疫器官组织病变现象，对大鼠血清及肝脏中IgA、IgM、IgG、IL-12及SOD指标的改善程度达到显著差异水平(P＜0.01)，对IL-2、TNF-α及GSH-Px及MDA指标的改善程度呈现较显著差异水平(P＜0.05)。（6）血清差异蛋白质组学研究结果表明新疆小白杏杏仁油可明显改变免疫力低下试验大鼠模型血清的蛋白表达谱。结合差异蛋白表达模式聚类的方法从251种差异表达蛋白中筛选获得了14种共性差异蛋白。采用KEGG Pathway数据库对14种共性差异蛋白进行作用通路富集，共获得其中10种共性差异蛋白的35条作用通路，免疫球蛋白重链V-III区(VH26)与其中20条通路均存在相关性；金黄色葡萄球菌感染、吞噬体、致病性大肠杆菌感染、原发性免疫缺陷、补体和凝血级联5条先天性免疫通路作用最为频繁，其相关作用共性差异蛋白包括结合珠蛋白(HP)、角蛋白(KRT10、KRT42)、微管蛋白α-8链(TUBA8)、免疫球蛋白k链V-III区(MOPC63)、α-1-抗蛋白酶(SERPINA1)及T-激肽原(KNG1)。（7）通过对血清剥夺-反应蛋白(SDPR)、T-合成蛋白Ⅰ亚基(CCT3)、N-乙酰-L-丙氨酸酰胺酶(PGLYRP2)及嗜铬粒蛋白-A(CHGA)4种未知Pathway共性差异表达蛋白的STRING互作分析，预测其与肿瘤与微生物感染抑制途径相关。（8）通过差异蛋白作用通路及生物活性物质生物学效应分析，再次论证了新疆小白杏杏仁油的抗氧化及免疫调节作用效果，其中不饱和脂肪酸(MUFA，PUFA)、β-谷甾醇△5、角鲨烯△2,6,10,14,18,22、α-生育酚等生物活性物质与14种共性差异蛋白之间存在可预见的多重相关性。本论文旨在通过新疆小白杏杏仁油主要生物活性物质指纹图谱及其应用研究寻找小白杏杏仁油种类和品质的鉴别与监控模式，并通过其生物学功能评价及作用分子机制研究深入明晰特色药食两用资源潜在功能价值。相关研究成果将为新疆小白杏资源功能性产品开发及品质认证提供良好的理论依据，加速新疆资源优势向经济优势的转化进程，对新疆特色优质资源的保护和产品品质升级具有深远的意义。更多还原

【Abstract】 Almond oil of small white apricot has been a common food and medicine resource inXinjiang for a long time. Traditionally, the Uygur residents in Xinjiang believe almond oilhave functions of nerves relief, anti-inflammatory, anti-mutagen and anti-bacteria. The currentissues, such as aging almond trees, endangering wild species and low degree of deepprocessing and comprehensive utilization, have limited development of almond oil industry inXinjiang. Although biological functions of special regional resources have drawn wideattention today, study on fingerprint of Xinjiang small white apricot almond oil and itsfunctions of antioxidant and immunomodulatory regulation is blank. The followinginvestigations were conducted based on small white apricot resources inventory in Xinjiang:Small white apricot almond oil from different areas in Xinjiang was extracted based onprevious researches to analyze the major biological active substances using GC-MStechnology and determine its fingerprint information; then, clustering and similarity analysiswere used to build the fingerprint library of bioactive substances. The varieties and quality ofsmall white apricot almond oil were identified and monitored with principal componentanalysis (PCA) and partial least squares discriminant analysis (PLS-DA); Small white apricotalmond from Kuche was taken as sample to undertake chromatographic studies for almond oilsamples prepared by different ways. The scavenging effects of different almond oil samples toDPPH, hydroxyl, superoxide anion, ABTS and other free radicals were investigated. Smallwhite apricot almond oil was composed by complex biological active substances. Dose-effectof antioxidant activity of almond oil was studied based on PLSR analysis;Immunocompromised rat model was established. Samples from supercritical carbon dioxideextraction during the study of antioxidant performance were prepared as base materials.Immunomodulatory effects of small white apricot almond oil were studied for blank feedinggroup (BS), model group (MC), model almond oil sample group (SF). Its immunomodulatoryeffects to immunocompromised rats were examined according to assessment of immune organindex, immune factors in serum (liver) and slices of immune tissues; Serum samples obtainedfrom rats of control group parallel to the immunomodulatory experiments were investigated.Differentially expressed proteins of serum were quantitatively identified through iTRAQlabeling and high performance liquid chromatography-electrospray ionization tandem massspectrometry (LC-ESI-MS/MS). Functional annotation to the differentially expressed proteinsidentified was conducted with expression pattern clustering (EPC) and metabolic pathway(Pathway) analysis to determine their most important biochemical metabolic pathways andsignal transduction pathways. The results and conclusions are as follows:(1)13model samples were screened from15basic almond oil samples using GC-MS and cluster analysis to develop fingerprint of characteristic compositions of small whiteapricot almond oil. Similarities and principle component analysis for fingerprint wereconducted for10randomly selected adulteration samples and other oil products. The resultsfully demonstrated that the proposed fingerprint was effective to assess quality of small whiteapricot almond oil samples (products). A qualitative discriminant model was developed fordifferent varieties of grease and almond oil based on PLS-DA. All correlation coefficients ofpredicted values and observed values were0.95or above. The samples were100%identified.This indicated that comprehensive use of fingerprint information and PLS-DA discriminantcan accurately detect and distinguish small white apricot almond oil and other grease.(2) Kuche small white apricot almond oil samples prepared by supercritical CO2extraction (SCDE), mechanical crushing extraction (MCE), ultrasonic assisted extraction(UAE) and Soxhlet extraction (SE) were analyzed using GC and GC-MS technologies. Totalof33kinds of common active substances screened, including fatty acid (19species), sterolsand terpenes (14species) showed significant differences (P﹤0.05, exclude α-tocopherol).The samples prepared by different methods presented different scavenging capacity to DPPH,hydroxyl, superoxide anion and ABTS. Samples prepared by SCDE and MCE showed thescavenging capacity to free radicals presented a trend to be stronger than VEcontrol group,especially to superoxide anion.(3) The correlation between samples prepared by different methods and antioxidantindicators were analyzed using PLS2model. Samples prepared by UAE presented relativelypoorer scavenging capacity to all of four kinds of free radicals. SCDE samples were stronglypositively correlated to oxidative stress indicators (DPPH radicals and hydroxyl radicals);compared with almond oil samples prepared by SCDE and MCE, SE samples showed positivecorrelations to all four antioxidant indicators, however, the correlations were relatively weak.(4) The correlations between specific active substances and antioxidant indicators wereanalyzed through PLS1model. Scavenging capacity to DPPH free radicals showedsignificantly positive correlations with cis-oleic acid△9, linoleic acid△9,12, linoleic acid△8,11,linolenic acid△6,9,12, eicosatrienoic acid△8,11,14, α(γ)-tocopherol, spinach sterol△7,22,β-sitosterol△5and squalene; scavenging capacity to hydroxyl free radicals had significantlypositive correlations with linoleic acid△9,12,△5-stigmasterol△5,22, β-sitosterol△5, γ-tocopheroland squalene; scavenging capacity to free radicals of superoxide anion showed significantlypositive correlations with palmitoleic acid, arachidonic acid△5,8,11,14, α-tocopherol,△5-avenasterol△5,24(28)and△7-avenasterol△7,24(28); scavenging capacity to ABTS freeradicals showed significantly positive correlations with trans-oleic acid△9, linoleic acid△9,12,linoleic acid△6,9,12, α-tocopherol,△5-avenasterol△5,24(28)and squalene. Squalene, linoleic acid△9,12and α-tocopherol presented significant correlations with three or more antioxidantindicators, which may be the important base materials. Squalene△2,6,10,14,18,22, arachidonic acid△5,8,11,14,△5-avenasterol△5,24(28)and linolenic acid△6,9,12may play the key synergism infree radical scavenging system.(5) It was found from immunocompromised rat model and Enzyme-linkedimmunosorbent assay that Xinjiang small white apricot almond oil can significantlystrengthen spleen index, thymus index and liver index of immunocompromised rat (P﹤0.05)and improve tissue structures of immune organs. Its improvements to IgA, IgM, IgG, IL-12and SOD of rat serum and liver were highly significant (P﹤0.01). For IL-2, TNF-α, GSH-Pxand MDA, the improvements were relatively significant (P﹤0.05).(6) Serum differential proteomics study results showed that Xinjiang small white apricotalmond oil can significantly change protein expression profile of immunocompromised rats.Total of14common differential proteins were screened from251varieties of differentiallyexpressed proteins through cluster analysis. Enrichment analysis was conducted for14varieties of common differential proteins based on KEGG Pathway database. Total of35ofpathways for10kinds of common differential proteins were obtained, of which V-IIII sectionof immunoglobulin heavy chain (VH26) were related with20pathways;5innate immunepathways, including interactions with staphylococcus aureus infection, phagosome,pathogenic E. coli infection, primary immunodeficiency, complement and blood coagulationwere the most frequently. The relevant common differential proteins included haptoglobinprotein (HP), keratin (KRT10, KRT42), tubulin α-8chain (TUBA8), V-III section ofimmunoglobulin k chain (MOPC63), α-1-antiprotease (SERPINA1) and T-prokinin (KNG1).(7) Through STRING interactive analysis to unknown pathways of four commondifferential proteins, including serum deprivation-reactive protein (SDPR), T-syntheticproteinⅠsubunit (CCT3), N-Acetyl-L-Alanine amidase (PGLYRP2) and chromogranin-A(CHGA), suppressor pathways associated with tumors and microbial infections werepredicted.(8) The antioxidant and immunomodulatory effects of small white apricot almond oilwere demonstrated again by the analysis of pathways about differential proteins andbiological effects about related active substances. There were some foreseeable multiplecorrelations among active substances, such as unsaturated fatty acids, β-sitosterol△5,squalene△2,6,10,14,18,22, α-tocopherols, and14common differential proteins.This study aims to the fingerprint of major bioactive substances of Xinjiang almond andits application to look for a species and quality identification and monitoring model. Thepotential values of this food and medicine resource were further investigated throughevaluation of its biological function and molecular mechanisms. The results provided a goodtheoretical basis for development of functional products and quality certification, which had important significance for accelerating process from resource advantages to economyadvantage, protection of local high quality resources and quality upgrade.更多还原