【作者】 苏东晓；

【导师】 张名位；

【作者基本信息】 华中农业大学 ， 农产品加工及贮藏工程， 2014， 博士

【摘要】 荔枝(Litchi chinensis Sonn.)是热带亚热带地区重要的代表性水果。我国荔枝种植面积和产量均居世界第一。由于荔枝采收期集中,且不耐储藏,精深加工成为推动荔枝产业健康、可持续发展的主要出路。作为岭南地区的代表性珍稀果品,荔枝自古被认为具有一定的滋补功效。探明其健康效应的物质基础和作用机理对于指引荔枝的精深加工方向有重要意义。虽然已经有研究从现代药理学角度报道了荔枝果肉的抗氧化、抗辐射和保护肝脏等功效,然而上述各项研究尚未涉及其活性物质基础。项目组及国内外同类研究结果表明荔枝果肉富含酚类物质,具有明显的抗氧化活性,但对果肉中各种单体酚类物质的活性差异还缺乏认识。大量研究表明多种不同来源的酚类物质对改善机体血脂代谢有明显作用。荔枝果肉多酚作为亚热带地区居民重要的膳食多酚来源对于脂质代谢的影响尚未见研究报道。为此,本研究在确定荔枝果肉多酚最佳提取条件的基础上,探讨其对高脂血症小鼠脂质代谢的调节作用,通过分析摄入荔枝果肉多酚对血脂代谢相关microRNA及其下游靶基因表达的影响,探明其调节脂代谢分子机制；进而在体外活性跟踪下鉴定出荔枝果肉主活性单体组分。研究结果对促进荔枝消费、指导荔枝功能食品精深加工、推动荔枝产业可持续发展具有重要意义。1.不同提取方法对荔枝果肉游离酚和结合酚及其抗氧化活性的影响：分别用不同极性5种溶剂提取荔枝果肉游离酚,采用酸水解法和碱水解法提取结合酚,采用氧自由基吸收能力分析(ORAC)法和细胞抗氧化分析(CAA)法测定其游离酚和结合酚的抗氧化活性。结果表明,荔枝果肉丙酮提取物游离酚含量最高。该方法提取后的果肉残渣分别采用酸水解法和碱水解法提取结合酚发现前者的提取效率是后者的2倍。不同溶剂提取的荔枝果肉游离酚中丙酮提取物的ORAC抗氧化活性最高。但丙酮与甲醇提取物的CAA抗氧化活性相当,高于乙醇和乙酸乙酯提取物,水提物CAA值最低。酸水解法得到的结合酚ORAC值和CAA值分别是碱法的2.6和1.9倍。上述结果表明丙酮水溶剂体系适于荔枝果肉游离酚提取,酸水解法较碱水解法适于提取荔枝果肉结合酚提取。2.荔枝果肉多酚大孔树脂分离工艺优化：比较11种不同极性大孔树脂对荔枝果肉酚类提取物中总酚和总黄酮的静态吸附和解吸性能,优化其最佳大孔树脂动态吸附和解吸工艺参数；通过HPLC方法对所得组分酚类物质种类及其含量变化进行分析。HPD-826大孔树脂分离纯化荔枝果肉总酚和总黄酮效果最好,其吸附和解吸工艺参数为：荔枝果肉酚类提取物上样浓度0.8mg/mL,上样速度3.0BV/h,95%乙醇溶液作为洗脱剂,洗脱流速3.0BV/h。经HPLC分析和鉴定,HPD-826分离纯化荔枝果肉酚类不会造成单体酚组成变化和明显损失；荔枝果肉酚类物质主要由3,4-二羟基苯甲酸、儿茶素、香草酸、咖啡酸、丁香酸、表儿茶素、高儿茶酚、阿魏酸和芦丁等9种单体组成,其中含量最高的依次是高儿茶酚、芦丁和表儿茶素,三者合计占到总量的94.37%。3.荔枝果肉多酚对高脂膳食小鼠的脂质代谢调控作用及其机制：以C57BL/6J雄性小鼠为研究对象,动物分为对照组,高脂模型组和荔枝多酚组。荔枝多酚组摄入高脂膳食的同时通过灌胃摄入剂量为500mg/kg.d的荔枝果肉多酚大孔树脂纯化提取物。10w后结束实验,酶法分析各组动物血清和肝脏中脂代谢相关指标,采用实时定量PCR分析荔枝果肉多酚对高脂膳食小鼠肝脏miR-33、miR-122和miR-370及其靶基因ABCA1、Fas和Cpt1等mRNA水平,并通过Western blotting分析ABCA1、Fas和Cpt1a等蛋白的表达情况。结果表明,荔枝多酚组较高脂组小鼠血清总甘油三酯和总胆固醇含量降低,高密度脂蛋白含量增加；摄入荔枝果肉多酚可以明显减轻高脂膳食诱导的肝脏脂肪变性；与高脂膳食模型组比较荔枝果肉多酚可降低小鼠肝脏miR-33和miR-122表达水平,下调其靶基因Fas的表达水平,上调ABCA1和Cptla的表达。提示荔枝果肉多酚通过调节miR-33和miR-122及其下游相关基因的表达水平,促进肝脏胆固醇外运和高密度胆固醇形成,同时降低脂肪酸合成并加速其氧化利用,从而发挥调节血脂的作用。4.荔枝果肉多酚的的结构鉴定及其细胞抗氧化活性：为进一步探明荔枝果肉主要活性酚类物质单体组成,本研究采用聚酰胺树脂对上述经HPD-826型大孔树脂分离的荔枝多酚提取物进行进一步纯化,并采用CAA和ORAC活性跟踪评价不同级分抗氧化活性,以分离出其主要抗氧化级分。对抗氧化活性高的级分经制备型反相液相色谱纯化获得3个单体组分,经ESI-MS、1H-NMR、13C-NMR和HMBC谱分别鉴定为槲皮素-3-O-素芸香糖-7-O-a-L-鼠李糖苷、芦丁和表儿茶素。此3种黄酮类化合物的含量依次为17.25mg/100g FW、3.58mg/100g FW和2.31mg/100g FW。HPLC鉴定的荔枝果肉中含量最高的高儿茶酚经波谱确证为槲皮素-3-O-芸香糖-7-O-a-L-鼠李糖苷。其显示出较好的CAA活性,与广泛报道的木屐草素相当,较桑色素和二氢杨梅素高。上述结果表明槲皮素-3-O-芸香糖-7-O-a-L-鼠李糖苷是荔枝果肉中含量最高,抗氧化活性最强的单体酚类成分。因此,该化合物可能是荔枝果肉发挥抗氧化及脂代谢调控等作用的主活性酚类单体。本研究的主要创新点：①建立了荔枝荔枝果肉中游离酚和结合酚的提取分离纯化方法,首次从荔枝中分离鉴定出酚类物质槲皮素-3-O-芸香糖-7-O-α-L-鼠李糖苷,并确证其是荔枝果肉多酚抗氧化作用的主活性组分。②发现并确证荔枝果肉多酚降低高脂血症小鼠的血脂水平并改善其血脂代谢作用,首次从其调节miR-33、miR-122和miR-370及其靶基因ABCAl、Fas、Cpt1a等mRNA阳蛋白表达水平变化的角度,明确了荔枝酚类物质调节脂代谢的分子机制。本研究鉴定的荔枝果肉酚类物质为揭示其健康效应提供了物质基础；从植物多酚调节miRNA及其靶基因mRNA表达水平变化的角度阐释荔枝果肉多酚调节血脂的生物活性,为其它果蔬的生物活性作用与分子机制研究提供了新方法和新思路。更多还原

【Abstract】 Litchi (Litchi chinensis Sonn.) is an important tropical to subtropical fruit. The litchiacreage and production of China rank first in the world. Since centralized harvest periodand intolerant storage of litchi, deep processing for promoting healthy and sustainabledevelopment of litchi industry is very important. Ancient records litchi has a tonic effect.To investgate the health effects material basis and bioactivity mechanism of litchi iscritical important to guide deep processing of litchi industry. Modern pharmacologicalstudies confirmed that litchi fruit has anti-oxidant anti-radioactive and hepatoprotectivefunctions. However, little is known on its bioactive substances. Our lab and abroadresearch showed similar results that litchi pulp is rich in polyphenols and has significantantioxidant activity. But still lack of awareness of the antioxidant activity of differentphenolics from litchi pulp. Modern Nutrition found that polyphenols regulate the miRNAexpression of lipid metabolism. As an important source of dietary polyphenols forsubtropical area residents the effects of litchi pulp polyphenols on lipid metabolism hasnot been reported. Therefore, the present study was to investigate the regulation lipidmolecular metabolism of litchi pulp phenolics in hyperlipidemic mice from the expressionlevels of miRNA after determine the optimum extraction conditions for litchi pulppolyphenols, to elucidate the major bioactive structure substances of phenolic compounds.The results have important implications for the promotion of litchi consumption,functional food deep processing and litchi industry sustainable development.1. Comparison of phenolic profiles extraction efficiency and cellular antioxidantactivities of litchi pulp extracts from different solvents Five different solvent mixtures were used to extract free phenolics from litchi pulp.Alkaline and acid hydrolysis were tested to hydrolysis bound phenolics from litchi pulpresidue. The antioxidant activities of litchi pulp extracts were evaluated using the oxygenradical absorbance capacity (ORAC) and cellular antioxidant activity (CAA) assays.Aqueous acetone extraction of litchi free phenolics exhibited the highest free phenolicscontent. The acid hydrolysis bound phenolics content of litchi pulp was2.0times higher than those obtained by alkaline hydrolysis. Aqueous acetone extraction exhibited the highest ORAC value. But for CAA, aqueous acetone extraction represented the same CAA value as aqueous methanol extraction. The bound phenolics obtained by acid hydrolysis exhibited2.6times ORAC and1.9times CAA values relative to that obtained by alkaline hydrolysis. Aqueous acetone solvent suitable for free phenolics extraction of litchi pulp, and acid hydrolysis exhibited better extraction efficency for its bound phenolics.2. Separation and purification of phenolic profiles in litchi pulp by macroporous resinThe static adsorption and desorption performance of eleven different polarities macroporous resins to litchi pulp total phenolics and total flavonoids were compared to select suitable resin for purification of phenolic compounds. The dynamic adsorption and desorption process parameters of macroporous resin of HPD-826were optimized. The phenolic compounds types and contents of litchi pulp were analyzed by HPLC. The results showed that HPD-826macroporous resin exhibited the best capability of adsorption and desorption of total phenolics and total flavonoids in litchi pulp. The optimal separating process parameters were as follows:the concentration of litchi pulp extract and the sampling rate were0.8mg/mL and3.0BV/h, respectively, and the elution concentration and flow velocity were95%ethanol and3.OB V/h, respectively. The contents of phenolic compounds of litchi pulp were deduced a little, but the phenolic profiles of litchi pulp were not changed after adsorption and desorption by HPD-826macroporous resin. Nine phenolic compounds,3,4dihydroxybenzoic acid, catechin, vanillic acid, caffeic acid, syringic acid, epicatechin,4-methylcatechol, ferulic acid and rutin, were preliminary identified by HPLC. The major phenolic profiles were4-methylcatechol, rutin and epicatechin. The percentage contribution of the three compounds to the total phenolic content was94.37%. In conclusion, HPD-826macroporous resin could be applied to purify total phenolics and total flavonoids in litchi pulp.3. Molecular mechanism of litchi pulp polyphenols hypolipidemic effectC57BL/6J male mice were divided into control group, high fat diet group and litchi pulp polyphenol group. Litchi pulp polyphenol group intake of high fat diet while intake by gavage a dose of500mg/kg?d of litchi pulp polyphenols separation and purification by macroporous resin. Ten weeks later, the levels of serum and liver lipid metabolism related indicators were analyzed by enzymatic assays. miRNA and mRNA were analyze using real-time quantitative PCR, and proteins were studied by Western blotting. Litchi pulp polyphenols represses miR-33and miR-122in the liver mice models that were induced by a high fat diet or a high fat diet plus dietary polyphenol extracts. Litchi pulp polyphenols promotes the expression of Abcal, miR-33target gene, and represses the expression of miR-122inderect target gene Fas. Besides, LPP improves fatty acid oxidation by enhance the expression of Cptla. These results show that LPP enhance the liver cholesterol efflux and improve HDL formation and reduce fatty acid synthesis and raise fatty acid oxidation. Therefore, the new molecular mechanism of LPP exert hypolipidemic effects in the liver could be considered the effects of LPP on the suppression of miR-33and miR-122.4. Structural elucidation and cellular antioxidant activity evaluation of major antioxidant phenolics in litchi pulpIn the present study, the major contributors to the antioxidant activity of fresh litchi pulp were identified and their cellular antioxidant activities were investigated. Aqueous acetone extracts of litchi pulp were fractionated on polyamide resin, and those fractions with the largest antioxidant and radical scavenging activities were selected using CAA and ORAC assays. Three compounds that were major contributors to the antioxidant activity in these fractions were obtained by reverse-phase preparative HPLC and identified as quercetin3-O-rutinoside-7-O-a-L-rhamnosidase (quercetin3-rut-7-rha), quercetin3-O-rutinoside (rutin) and (-)-epicatechin using NMR spectroscopy, HMBC, and ESI-MS spectrometry. The concentration of these three compounds were17.25,3.58and2.31mg per100g of litchi pulp fresh weight. The highest content of litchi fruit4-methylcatechol identified by HPLC was confirmed as quercetin3-rut-7-rha by spectroscopic. These results suggest that the highest content and strongest antioxidant activity component of litchi pulp was quercetin3-rut-7-rha. Thus, this compound of litchi pulp may exert the effects of antioxidant and regulation of lipid metabolism. This is the first report of the identification and cellular antioxidant activity of quercetin3-rut-7-rha from litchi pulp.The highlights of the present study could be summarized as following two points. Firstly, extraction and purification methods of free and bound phenolics of litchi pulp were established. The phenolic compounds quercetin-3-O-rutinose-7-O-a-L-rhamnoside was fistly isolated and identified from litchi pulp, and confirmed its as the major antioxidant component which could provide the material foundation to reveal the health effects of litchi. Secondly, Discovered and confirmed polyphenols of litchi pulp exert hypolipidemic effects and improve lipid metabolism. For the first time clear molecular mechanisms of litchi phenolics regulating lipid metabolism from the expression of miR-33, miR-122and miR-370and its target gene ABCA1, Fas, Cptla and protein.The topic of the present study elucidated the structural of major antioxidant phenolics in litchi pulp which could provides a scientific basis to reveal the health. protective effect of polyphenols structure-activity relationships.This study investgated the effects of plant polyphenol on the expression of miRNA and its target genes mRNA of lipid metabolism. This could provide new methods and new ideas to explore the molecular mechanisms of other fruits and vegetables biological activity.更多还原