【作者】 孙怡；

【导师】 胡秋辉；

【作者基本信息】 南京农业大学 ， 食品科学， 2010， 博士

【摘要】 苦丁茶是一种纯天然植物代用茶,富含三萜及其皂苷类、黄酮、氨基酸等活性物质,具有抗氧化、降脂、抗炎杀菌、减肥、降血压等多种药理功能。在我国,主要的苦丁茶是利用冬青科树叶加工而成的苦丁茶和利用木犀科树叶加工而成的苦丁茶,而前者主要有苦丁茶冬青和大叶冬青。近年来,已有人对冬青苦丁茶的化学成分和药理作用作了一些研究,但研究多集中在粗提物药理作用及其机制的研究上,化学成分研究仅限于三萜及其皂苷类、黄酮类的研究。因此,本研究以苦丁茶冬青苦丁茶和大叶冬青苦丁茶(两者通称为冬青苦丁茶)为原料,较为系统地研究其多酚类物质的测定方法、提取、纯化、结构鉴定和抗氧化活性,同时采取热水提取与醇沉制备冬青苦丁茶多糖,利用层析技术对其进行分离纯化,并对冬青苦丁茶粗多糖及其纯化组分的基本理化性质、体外抗氧化活性进行分析。1.冬青苦丁茶多酚含量的测定及其抗氧化活性以绿原酸为标准品,建立了测定冬青苦丁茶中多酚含量的Folin-Ciocalteu比色法。结果表明,苦丁茶提取液在Folin-Ciocalteu试剂3.0 ml、饱和Na2CO3溶液4.5ml、反应温度30℃、反应时间30 min的条件下,测定其747nm处的吸光值,多酚浓度在20.0～100.0 mg/L范围内与吸光值呈良好的线性关系；稳定性、精密度、重现性和回收率实验的相对标准偏差为0.887～1.416%；与酒石酸亚铁比色法的测定结果无显著差异。苦丁茶冬青苦丁茶热水提取物(粗提物)经不同的有机溶剂分部萃取,得到氯仿萃取物、乙酸乙酯萃取物、正丁醇萃取物及萃取剩余物,然后采用Folin-Ciocalteu比色法测定粗提物和各萃取物的多酚含量,同时应用DPPH法、TEAC法和FRAP法分别测定粗提物和各萃取物的自由基清除能力和还原Fe3+能力。结果表明,苦丁茶提取物具有较高的多酚含量和较强的抗氧化能力,DPPH法和FRAP法测定各提取物抗氧化能力的结果为乙酸乙酯萃取物>正丁醇萃取物>粗提物>萃取剩余物>氯仿萃取物,TEAC法测定结果为乙酸乙酯萃取物>正丁醇萃取物>粗提物>氯仿萃取物>萃取剩余物；多酚含量与抗氧化能力之间、所用抗氧化测定方法之间均存在较好的相关性。比较了苦丁茶冬青苦丁茶和大叶冬青苦丁茶提取液中多酚、黄酮的含量及体外抗氧化能力,结果表明多酚含量较高的苦丁茶冬青苦丁茶比黄酮含量高的大叶冬青苦丁茶具有较高的体外抗氧化能力。对五种体外抗氧化活性评价方法之间的相关性进行了分析,结果表明各抗氧化方法间相关性良好(R>0.8478),尤以DPPH法与ABTS法的相关性最高(R 0.9967)。2.冬青苦丁茶多酚提取条件的优化提取溶剂甲醇、乙醇和水对苦丁茶多酚的提取有显著的影响,提取物的多酚含量随提取溶剂的不同而不同,甲醇、乙醇和水三者之间提取物多酚含量差异显著(P<0.05),这三者中乙醇的提取效果最好。选择乙醇浓度、提取温度和提取时间3个因素分别进行实验,比较提取物的多酚含量,确定了各因素在CCD实验中的取值中心点,即乙醇浓度、提取温度和提取时间分别是70%、70℃和60 min。在单因素实验基础上,以提取物的多酚含量和清除DPPH自由基能力IC50值为响应值,利用Design-Expert软件对乙醇浓度、提取温度及提取时间3个参数进行三因素三水平的CCD实验,对3个参数的实验组合进行优化。获得的最优提取条件为：乙醇浓度70.5%、提取温度73.9℃和提取时间61.2 mmin的条件下可以得到最高的多酚含量(235.24 mg CHA/gDW);乙醇浓度70.2%、提取温度70.6℃和提取时间60.4 min时可以获得最大的DPPH自由基清除能力(IC50,480.87μg/mL)。可以看出,两组优化条件基本一致,说明可以用相同的提取条件获得最优的多酚含量和抗氧化能力的苦丁茶提取物。用水为提取溶剂时,提取温度和提取时间2个因素对苦丁茶多酚的提取有显著的影响。通过实验优化,得到了苦丁茶多酚的最优提取条件为：提取温度91.2℃和时间36.1 min,提取物总酚含量达198.45 mg CHA/gDW;提取温度92.2℃和35.7 min,提取物清除DPPH自由基能力最强(IC50,744.74μg/mL)。同样,两组优化条件基本一致,说明以水为提取溶剂时可以用相同的提取条件获得最优的总多酚含量和抗氧化能力的苦丁茶提取物。选用5次优化的提取条件和5次随机设定的提取条件,对模型方程的适用性进行验证。结果表明,验证性实验的实验值与模型方程的预测值间存在很好的一致性,说明优化出的提取条件是切实可行的。3.苦丁茶冬青苦丁茶中多酚的分离纯化与结构解析利用HPLC-DAD分析了冬青苦丁茶、女贞苦丁茶与绿茶(雨花茶)的多酚类化合物组成,结果表明它们的色谱峰图呈现明显的不一致,这说明苦丁茶的主要化学成分可能不同于绿茶,苦丁茶冬青苦丁茶和女贞苦丁茶的组成可能也不一样。通过HPLC-MS知HPLC-DAD分析,结合茶叶儿茶素[(-)-EC、(-)-ECG、(-)-EGC知(-)-EGCG]、没食子酸、咖啡碱、茶叶碱和可可碱的标准样叠加分析验证,绿茶的主要多酚类成分为没食子酸、没食子酰基奎尼酸、EC、(-)-EGC、(-)-EGCG。根据苦丁茶冬青苦丁茶的HPLC-MS分析结果与相关参考文献,推测其主要多酚类化合物为绿原酸及其衍生物,即3-咖啡酰基奎尼酸、绿原酸、4-咖啡酰基奎尼酸、3,4-二咖啡酰基奎尼酸、3,5-二咖啡酰基奎尼酸和4,5-二咖啡酰基奎尼酸。而根据女贞苦丁茶的HPLC-MS分析结果,认为女贞苦丁茶的主要酚类化合物为苯乙醇苷类成分,即紫茎女贞苷A、Ligurobustoside N、紫茎女贞苷C、紫茎女贞苷B和Osmanthuside B6。利用半制备型HPLC从苦丁茶冬青苦丁茶中分离纯化得到5种单体化合物,通过MS和NMR分析结合]3PLC-DAD、HPLC-MS的分析结果,将5种单体化合物分别鉴定为羟基酪醇葡萄糖苷(hydroxytyrosol glucoside)、绿原酸、4,5-0-二咖啡酰奎尼酸、3,5-O-二咖啡酰奎尼酸和3,4-O-二咖啡酰奎尼酸,其中化合物1是首次从苦丁茶冬青苦丁茶中分离得到。4.大叶冬青苦丁茶多糖提取、纯化与抗氧化活性大叶冬青苦丁茶经热水提取、脱色、醇沉、干燥,得到大叶冬青苦丁茶粗多糖(ILPS).粗ILPS经DEAE-52纤维素层析柱分离得到四个多糖组分,即ILPS-1、ILPS-2、ILPS-3知ILPS-4.红外光谱分析表明粗ILPS及其纯化组分均是多糖类物质,粗ILPS与各纯化组分的蛋白质、糖醛酸和硫酸基含量存在很大的差异,粗ILPS. ILPS-1、ILPS-2知ILPS-3主要由鼠李糖、阿拉伯糖和半乳糖等组成。采用化学法分别测定了粗ILPS及其纯化组分的体外清除自由基(DPPH-O2-、·OH)能力、还原能力以及螯合金属能力。结果表明,大叶冬青苦丁茶多糖具有较强的体外抗氧化活性,并且抗氧化能力与多糖浓度之间存在良好的相关性。更多还原

【Abstract】 Kudingchahas, one kind of herbal tea, is rich in alkaloid, flavonoids, and amino acids, resulting in many beneficial functions such as cardiovascular, antioxidant, antiobesity, antidiabetic, anti-inflammatory, neuroprotective and hepatoprotective effects. In China, the most common kudingcha categories consumed are from 2 genera in two families, i.e., the genus Ilex in the family Aquifoliaceae and the genus Ligustrum in the family Oleaceae. In genus Ilex, the leaves of Ilex kudingcha and Ilex latifolia have often been used as the materials for production of kudingcha. Recently, there are some reports about its chemical compositions and pharmaceutical functions. However, much attention has been paid on the biological functions, mechanism and some bioactive compounds such as flavonoids, triterpenes and triterpenoid saponins in crude extracts. Therefore, the first purpose of the present work was to study the determination, extraction, purification, identification and antioxidant activity of polyphenols in kudingcha made from Ilex kudingcha C.J. Tseng or Ilex latifolia. The second one was to investigate the extraction, purification, preliminary characterization and antioxidant activity of polysaccharides of kudingcha made from I. latifolia.1. Determination of the total polyphenol content and antioxidant activity of I. kudingchaA quantitative method for the determination of the total content of polyphenols (TPC) in kudincha was studied by Folin-Ciocalteu colorimetry (FC method) with chlorogenic acid as standard. The results showed that the TPC of kudincha could be well calculated according to their colorimetric absorption at 747 nm by applying Folin-Ciocalteu reagent 3.0 ml and saturated Na2CO34.5 ml at 30℃for 30 min, and the linear range of standard curve was 20.0～100.0 mg/L. Stability, precision, repetition and recovery rate were in the range of 0.887～1.416% by RSD (relative standard deviation). There are not any differences for the results obtained by FC method and ferrous tartrate colourimetry.The hot water extract (crude extract) of kudingcha made from I. kudincha C.J. Tseng and its four fractions of chloroform, ethyl acetate, n-butanol, and water were prepared. The TPC and the antioxidant activities of the extracts were determined by Folin-Ciocalteu method and DPPH assay, FRAP assay, and TEAC assay, respectively. The results showed that kudingcha extracts contained high TPC and showed potent antioxidant activity. The antioxidant activities of crude extract and its fractions decreased in the order of ethyl acetate fraction>butanol fraction> crude extract> water fraction> chloroform fraction according to the DPPH assay and FRAP assay, which were the same with the exception of the rank order of water fraction and chloroform fraction obtained from TEAC assay. Satisfactory correlations between TPC and antioxidant activity and between measuring methods of antioxidant activity were observed. It suggests that the polyphenols in the extracts are largely responsible for the antioxidant activities.The contents of polyphenol and flavonoid and the antioxidant activities in vitro of kudingcha crude extracts between I. latifolia and I. kudingcha C.J. Tseng were determined. The results showed that the crude extract of kudingcha from I. kudingcha C.J. Tseng with higher TPC showed higher antioxidant activity than that of I, latifolia with higher flavonoid content. In addition, the correlations between evaluation methods for antioxidant activities were good (R>0.8478), especially that between DPPH assay and ABTS assay (R 0.9967).2. Optimization of extraction conditions for phenol antioxidants from kudingchaThe TPC of extract varied greatly with the solvents used (methanol, ethanol and water). Statistical analysis showed that significant differences were existed among ethanol, methanol and water (P<0.05) as solvent. It has demonstrated that ethanol solution was the most effective in extracting phenolic compounds from kudingcha.The effects of three factors of ethanol concentration, extraction temperature and time on the extraction of polyphenols were investigated to determine the appropriate experimental ranges for central composite design. As results, ethanol concentration 70%, extraction temperature 70℃and extraction time 60 min were chosen as the central points. Based on results of single-factor experiments, Design-Expert 7.1.3 software was then employed to determine the optimal combination of extraction variables for the extraction of polyphenolic antioxidants with the total polyphenol content and IC50 of scavenging activity on DPPH free radical as the response values. Through optimization, the optimal extraction conditions with ethanol solution as solvent were as follows:ethanol concentration 70.5%, extraction temperature 73.9℃and time 61.2 min for TPC (235.24 mg chlorogenic acid/g); ethanol concentration 70.2%, extraction temperature 70.6℃and time 60.4 min for potent scavenging activity on DPPH free radical (IC50480.87μg/mL). While for water as a solvent, the optimal conditions were extraction temperature 91.2℃and time 35.7 min for TPC (198.45 mg chlorogenic acid/g) and extraction temperature 92.2℃and time 35.7 min for potent DPPH free radical scavenging activity (744.74μg/mL). Similar parameters were obtained for extraction with higher TPC and lower IC50 of scavenging activity on DPPH free radical when using either ethanol or water as solvent. The results suggested that the same optimal conditions could be used to get kudingcha extract with higher TPC and lower IC50 value of scavenging activity on DPPH free radical with ethanol or water as solvent.In order to validate the adequacy of the model equations, ten verification experiments were carried out under various extraction conditions (five of which were the optimized conditions, while the other five were randomly chosen). The results indicated that the experimental values were in good agreement with the predicted ones, and also suggested that the regression models were accurate and adequate for the extraction of polyphenol antioxidants from kudingcha.3. Isolation and structure elucidation of phenolic compounds in kudingchaThe polyphenolic substances in kudincha and green tea (Yuhua greent tea) were firstly analyzed by high-performance liquid chromatography with diode-array detector (HPLC-DAD). It was found that the polyphenolic substances in kudincha might be quite different from those in green tea made from Camellia sinensis L. In addition, the polyphenolic substances in kudincha made from I. Kudincha C.J. Tseng might be quite different quite from those in kudingcha made from Oleaceae.The main phenolic compounds in green tea were gallic acid, galloylquinic acid, EC, EGC, ECG and EGCG through the analysis of HPLC-DAD and HPLC-mass spectroscopy combined with verification experiments of standard compounds in tea. In similar manner, caffeoylquinic acid (CQA) derivatives such as 3-CQA,4-CQA,5-CQA (chlorogenic acid), 3,4-diCQA,3,5-diCQA, and 4,5-diCQA were suggested for the main phenolic compounds of kudingcha made from I. Kudincha C.J. Tseng, while ligupurpuroside A, ligurobustoside N, ligupurpuroside B, ligupurpuroside B and osmanthuside B6 for kudingcha made from Oleaceae.Five compounds were isolated from the extract of kudingcha made from I. Kudincha C.J. Tseng by semi-preparative HPLC. They were characterized as hydroxytyrosol glucoside,5-CQA,3,4-diCQA,3,5-diCQA, and 4,5-diCQA, respectively, by HPLC-DAD, HPLC-MS and NMR.4. Extraction, purification and antioxidant activity of polysaccharides from kudingcha made from I. latifoliaKudingcha made from I. latifolia was defatted with 85% ethanol, and then extracted with hot water, decolored by S-8 macroporous resin, precipitated by ethanol, and dried to afford crude polysaccharide (ILPS). The crude ILPS was purified by DEAE-52 cellulose anion-exchange chromatography, resulting in four purified polysaccharides as ILPS-1, ILPS-2, ILPS-3 and ILPS-4. Furthermore, the crude and purified ILPS were preliminary characterized by GC and FT-IR. As results, it has been demonstrated that the crude ILPS and its purified fractions were polysaccharides by FT-IR analysis. Differences in the contents of protein, uronic acid and sulfate were found for crude ILPS and its purified fractions. And rhamnose, arabinose and galactose were found the main monosaccharide compositions for crudeILPS, ILPS-1, ILPS-2 and ILPS-3. Finally, the antioxidant activities in vitro of the crude ILPS and its purified fractions were evaluated by determinations of scavenging activities on DPPH free radicals, superoxide anion radicals (O2.), hydroxyl radicals (.OH), ferric reducing-antioxidant power and chelating capacity. The results indicated that ILPS exhibited a strong antioxidant activity, and the antioxidant activity was significantly correlated with its concentration.更多还原