【作者】 李红艳；

【导师】 邓泽元； 曹荣；

【作者基本信息】 南昌大学 ， 营养与食品卫生学， 2012， 博士

【摘要】 研究表明,番茄中的植物化学物对人体有抗氧化、延缓心血管疾病及抗癌等保护作用。植物化学物的快速定性定量分析对番茄品种的选育、番茄制品的加工过程监测及品质评价具有重要的意义。国内外研究表明,番茄中的主要植物化学物如类胡萝卜素的不同顺式异构体结构类似,而酚类化合物多以复杂糖苷的形式存在。因此,对它们的分离、鉴定存在着耗时较长、工艺复杂、鉴定困难等问题。目前,对番茄抗氧化能力的测定方法不一,没有系统评估、对比其抗氧化活性的方法体系。本课题针对不同品种番茄中的多种植物化学物如类胡萝卜素、酚类化合物和紫色番茄中特有的花青素分别进行提取、分离及结鉴定。建立了快速、准确、高效分离鉴定植物化学物的高效UPLC方法；将微波辅助提取技术结合响应曲面法用于优化番茄中酚类化合物的最佳抗氧化能力的提取工艺；采用快速、准确的多种96孔板微型评价方法(DPPH、PCL、FRAP、ORAC)研究了不同番茄溶解性不同的提取物(脂溶性和水溶性)的体外抗氧化活性；模拟体内自由基对心肌细胞的氧化损伤,探讨其在H202诱导下,对大鼠心肌H9c2细胞损伤及凋亡的保护作用；评价了紫色番茄V118的细胞内抗氧化及抗癌能力。本研究从本质上探讨了不同番茄中植物化学物的组成、含量与其抗氧化、抗癌活性之间的内在关系。本课题研究的最终目标为指导、培育和开发不同植物化学物含量、抗氧化活性及符合不同消费者营养需求的优质番茄品种。本实验依据以上实验内容,得到如下结果：1.建立了快速、准确测定番茄中不同顺、反式类胡萝卜素结构包括叶黄素、番茄红素和β-胡萝卜素的UPLC方法,在15min内分离鉴定了3个全反式和21个不同顺式结构的类胡萝卜素。其中顺式类胡萝卜素占总类胡萝卜素含量的10%左右,主要包括9-、13-cis-叶黄素,5-、9-、13-、15-cis-番茄红素和9-、13-、15-cis-β-胡萝卜素。2.以抗氧化活性(FRAC和ORAC法)为指标,将微波辅助提取技术结合响应曲面法用于番茄中不同酚类化合物的提取,优化了微波辅助提取工艺的各项技术参数。以FRAP值最佳抗氧化活性为指标时,优化的微波辅助提取条件为：微波温度96.5℃,提取时间2.06min,乙醇浓度66.2%；以ORAC值最佳抗氧化活性为指标时,优化的微波辅助提取条件为：微波温度96.5℃,提取时间1.66min,乙醇浓度61.1%。其中微波温度和乙醇浓度对提取过程的影响较大。结果表明,微波辅助提取具有提取时间短、选择性好、提取率高、溶剂消耗量小等优点。3.建立了9min内快速、准确测定20个不同颜色不同品种番茄中13个酚类化合物的UPLC方法,其中主要的酚类化合物为绿原酸、对香豆酸、龙胆酸、阿魏酸、咖啡酸、原儿茶酸、芦丁和柚皮素。4.分离、鉴定了新型紫色番茄品种V118中的植物化学物组成。其中总类胡萝卜素含量为234.78μg/g DW,总酚含量为659.11mg GAE/100g DW。对紫色番茄中特有的花青素进行定性、定量分析。紫色番茄V118中三个主要的花青素分别为牵牛色素-3-O-咖啡酰基-芦丁糖苷-5-O-葡萄糖苷(petunidin-3-O-caffeoyl-rutinoside-5-O-glucoside)、牵牛色素-3-O-对香豆酰基-芦丁糖苷-5-O-葡萄糖苷(petunidin-3-O-(p-coumaryl)-rutinoside-5-O-glucoside)和锦葵色素-3-O-对香豆酸-芦丁糖苷-5-O-葡萄糖苷(malvidin-3-O-(p-coumaryl)-rutinoside-5-O-glucoside),含量分别为9.04,50.18和13.09mg/100g DW(分别表示为牵牛色素和锦葵色素当量)。5.在相同的种植环境、栽培条件和田间管理技术下,不同品种番茄中的植物化学物含量之间表现出显著性差异,说明基因可能是影响番茄中植物化学物组成和含量的主要因素之一。不同的番茄品种中,O258AAAA的总类胡萝卜素含量最高(261.86μg/gDW),其次为Q105AAAA (242.02μg/g DW)和LA1593(232.12μg/g DW)。总得来说,红色番茄中的总类胡萝卜素含量最高,其次为紫色、橙色、粉色和黄色。番茄红素是番茄中含量最高的类胡萝卜素(9.61-227.11μg/g DW),其次为p-胡萝卜素(6.89-110.40μg/g DW)和叶黄素(2.85-9.23μg/gDW)。 Q105AAAA的总酚含量最高,为997.45mg GAE/100g DW,其次为O258AAAA和H9478,分别为883.72mg GAE/100g DW和746.09mg GAE/100gDW。番茄中单个酚类化合物的含量为6.10-42.73mg/100g DW,其中芦丁和绿原酸的含量最高。6.番茄脂溶性和水溶性提取物均有较强的抗氧化活性。其中O258AAAA和Q105AAAA的抗氧化活性最高。20种不同品种番茄脂溶性提取物的PCL抗氧化活性为6.01-48.83μmolTE/g DW, DPPH法测定的ECso值为7.76-56.98μg；水溶性提取物的FRAP值为26.86-57.84μmol AAE/g DW, ORAC值为235.42-351.19μmol TE/g DW。 V118紫色番茄的脂溶性提取物的PCL值为30.]1μmol TE/g DW, ORAC-L值为11.97μmol TE/g DW;水溶性提取物FRAP值为54.95μmol AAE/g DW, ORAC值为323.23μmol TE/g DW。紫色番茄V118在保持传统番茄原有的植物化学物的前提下,增加了花青素的含量,提高了其抗氧化活性。7.番茄中脂溶性提取物的PCL和DPPH抗氧化活性与总类胡萝卜素含量呈显著正相关(p<0.05),水溶性提取物的FRAP抗氧化活性与总酚含量和总酚含量指数呈显著正相关(p<0.05)。说明番茄中的植物化学物含量与其抗氧化活性之间有密切相关性。8.由类胡萝卜素含量不同而引起的不同颜色差异的番茄品种,其脂溶性提取物对H202诱导的大鼠心肌H9c2细胞损伤及凋亡的保护作用有明显差别。不同番茄品种的总类胡萝卜素含量、抗氧化活性和保护细胞作用从大到小依次为：红、紫、橙、粉、黄。抗氧化保护机制主要是通过抑制半胱天冬酶-3和基质金属蛋白酶-2的活性体现,且都与番茄红素的含量呈显著正相关(p<0.05)。番茄红素抑制H202溶液引起细胞损伤及凋亡的能力优于叶黄素和p-胡萝卜素,番茄提取液的抑制作用优于单个番茄红素、叶黄素或β-胡萝卜素标准品溶液。说明番茄中不同植物化学物之间的抗氧化能力有相互增效作用。9.紫色番茄中不同的植物化学物能被乳腺细胞MCF-10A吸收,并且在细胞内表现出良好的抗氧化能力。采用不同体内消化酶处理紫色番茄中的植物化学物,模拟体外消化实验。结果表明：消化前后不同紫色番茄的脂溶性和水溶性提取物(类胡萝卜素和酚类化合物)能保护H202所引起的乳腺细胞损伤或凋亡,且其保护作用在正常乳腺细胞MCF-10A中优于乳腺癌细胞MCF-7。说明紫色番茄植物化学物有保护正常乳腺细胞并抑制乳腺癌细胞生成的作用。10.紫色和红色番茄中的植物化学物能抑制大鼠足肿胀,说明它们有很强的抗炎能力。在总植物化学物含量相等的情况下,紫色番茄的体内抗氧化能力优于红色番茄,可能与其所含的花青素有关。分别交叉混合紫色和红色番茄中不同的脂溶性和水溶性提取物,发现含花青素的混合物抗炎能力更好。更多还原

【Abstract】 Epidemiological and experimental studies have shown that intake of tomato naturally high in phytochemicals is positively associated with health benefits, particularly in reducing risks of many prevalent chronic diseases such as cancer and coronary heart disease.The objectives of the present investigation are to:develop rapid and sensitive UPLC methods to obtain complete, pictures of phytochemicals ineclading carotenoids, phenolics in tomatoes; examine the antioxidant activities of carotenoid and phenolic extracts of20tomato cultivars and breeding lines using the chemical based mthods2,2-diphenyl-l-picrylhydrazyl (DPPH) free radical scavenging capacity; photochemiluminescence (PCL); ferric reducing antioxidant power (FRAP); oxygen radical absorption capacity (ORAC) assays and a cell-based model system using cardiac H9c2cells and H2O2, respectively; and assess the relationships between the antioxidant activity and phytochemical concentration and composition of tomatoes. The information of phytochemical contents and antioxidant activities of different tomato cultivars and breeding lines and their correlations will be useful to plant breeders, food processers and other food industry. Our ultimate goal is to breed designer tomatoes. The main results are shown as followed:1. All-trans lutein, lycopene, β-carotene and their21cis isomers in20tomato breeding were separated and identified by a rapid and sensitive UPLC method using a1.7μm C18column in15min. All-trans carotenoids were predominant, but9-cis,13-cis-lutein,5-cis,9-cis,13-cis,15-cis-lycopene,9-cis,3-cis and15-cis-β-carotene were also found.2. Microwave-assisted extraction (MAE) was investigated for extraction of phenolic compounds from tomato with maximized antioxidant activities using response surface methodology (RSM) coupled with a central composite design, and in vitro antioxidant assays (FRAP and ORAC). MAE was more efficient for greater antioxidant activities and higher total phenolic contents than solvent extraction. The optimal MAE processing parameters were96.5℃,2.06min,66.2%ethanol for FRAP, and96.5℃,1.66min,61.1%ethanol for ORAC.3. The phytochemical content was significantly different among the20^tomato cultivars. The discrepancy is not surprising because genotype can play an important role in the carotenoid and phenolic profiles of tomato. The highest total carotenoid content was found in O258AAAA (261.86μg/g DW), followed by Q105AAAA (242.02μg/g DW) and LA1593(232.12μg/g DW). Red tomatoes had the highest total carotenoid contents, followed by purple, orange, pink and yellow ones. For the individual carotenoids, lycopene had the highest concentrations (total of all-trans and cis isomers) ranging from9.61to227.11μg/g DW, followed by total β-ca(?)ene (6.89to110.40μg/g DW) and total lutein (2.85to9.23μg/g DW). The highest total carotenoid content was found in Q105AAAA (997.45mg GAE/100g DW), followed by O258AAAA (883.72mg GAE/100g DW) and H9478(746.09mg GAE/100g DW). Individual phenolic compounds of the20tomato varieties were ranging from6.10to42.73mg/100g.4. A newly developed non-genetically modified purple tomato V118was investigated for its phytochemical compositions and antioxidant activities. A highly efficient and sensitive UPLC method was developed for both the phenolics and carotenoids. The main carotenoids were lutein, lycopene and β-carotene while the main phenolics were protocatechuic acid, chlorogenic acid, gentistic acid, caffeic acid, p-coumaric acid, ferulic acid, rutin and naringenin. The total carotenoid content of V118was234.78μg/g DW and the total phenolic content was659.11mg GAE/100g DW.5. It showed that in addition to the phytochemicals commonly known for tomatoes, V118had a unique composition of anthocyanins. The LC-MS study showed three major anthocyanins which were petunidin-3-O-caffeoyl-rutinoside-5-O-glucoside (9.04mg petunidin equivalent/100g DW), petunidin-3-O-(p-coumaryl)-rutinoside-5-O-glucoside (50.18mg petunidin equivalent/100g DW) and malvidin-3-O-(p-coumaryl)-rutinoside-5-O-glucoside (13.09mg malvidin equivalent/100g DW).6. The extracts of different tomatoes showed strong but different antioxidant activity. O258AAAA and Q105AAAA exhibited the highest antioxidant activity. The antioxidant activities of the carotenoid extracts of all cultivars tested in the PCL assay ranged from6.01-48.83nmol Trolox equivalent/g DW, and the EC50values in the DPPH assay were from7.76-56.98μg. The FRAP values varied from26.86to57.84μmol AAE/g DW, while the ORAC values ranged from235.42to351.19umol TE/g DW. For purple tomato V118, the antioxidant activities of the lipophilic extract as measured by the PCL and ORAC-L assays were30.11μmol TE/g DW and11.97μmol TE/g DW, respectively; while the hydrophilic extracts as determined by the ORAC-H and FRAP assays were323.23μmol TE/g DW and54.95μmol AAE/g DW, respectively. It showed that purple tomatoes such as V118possess additional phytochemicals like anthocyanins which can potentially have added health benefits.7. The total antioxidant activities as evaluated by PCL and DPPH assays were found to correlate well with the total carotenoid content. The FRAP, but not the ORAC value showed good correlation with the TPC or TPI (p<0.05).8. The lipophilic extracts of pink, red, purple, orange and yellow tomatoes were found to prevent cell death in a cell-based model system using cardiac H9c2cells and H2O2, via attenuation of the caspase-3and matrix metalloproteinase (MMP)-2activities. In both chemical and cell based systems, extracts of different tomatoes showed strong but different antioxidant activities. Lycopene showed the highest correlation in the antioxidant activity.9. Purple tomato showed better chemical-based antioxidant activities, and the phytochemicals in V118can be transported across the MCF cell barrier and showed strong CAA.10. The phytochemicals in purple and red tomatoes had strong anti-inflammatory activity and could significantly inhibite paw oedema formation in a paw oedema rat model. The in vivo antioxidant and anti-inflammatory acitivity of purple tomato was much higher than red one because there was anthocyanin existed in the purple tomato.更多还原