【作者】 杨会成；

【导师】 曾名湧；

【作者基本信息】 中国海洋大学 ， 食品科学， 2008， 硕士

【摘要】 植物多酚与人类的生活息息相关,作为一类具有多种生物活性的天然物质,它对人的消化、营养、健康产生影响,已在食品、药品、化妆品等工业中得到了广泛的应用。本论文以鲜海带(Laminaria japonica Aresch)为原料,系统地研究了海带多酚(KP)的制备、分离纯化、抗肿瘤、抑菌活性,并初步探讨了其理化性质和作用机理,为深入研究海藻多酚提供了基础数据,同时也为海带的高值化利用及新的抗肿瘤药物、食品保鲜剂的开发提供一种新的思路。本论文的研究内容包括以下6个部分:1.海带多酚提取率测定方法的选择:海带可以综合利用提取多酚和其它副产物。通过酒石酸铁比色法和Folin-Denis法两种多酚定量分析方法的比较,表明以没食子酸丙酯为参照物的酒石酸铁比色法方便、快速、可靠,可以作为本次实验定量分析手段。另外,在同样的提取条件下,鲜海带总酚含量要比干海带的总酚含量略高。2.海带多酚提取工艺条件的优化:采用鲜海带为原料,以超声波、微波复合浸提为前处理手段,以多酚的提取率为指标,在单因素实验的基础上采用正交实验对KP浸提工艺参数进行了优化,结果表明:采用料液比1:7(g/mL),乙醇浓度85%,浸提温度70℃,浸提次数2次,浸提时间4h,提取效果最佳,KP提取率为2.08%。3.海带多酚粗提物抗肿瘤、抗菌活性的研究:以MTT法分别测定了KP粗提物对人肺腺癌A549细胞、鼠白血病P388细胞、人肝癌BEL-7402细胞和人宫颈癌Hela细胞的抗肿瘤活性;以平板打孔抑制法分别测定了KP粗提物对金黄色葡萄球菌、枯草芽孢杆菌、大肠杆菌、绿脓杆菌、痢疾志贺菌、产气肠杆菌、普通变形杆菌、白色念珠菌、青霉、灰霉、黄曲霉的抑菌活性。结果表明:KP粗提物具有一定的抗肿瘤活性,在100μg/mL时,对A549、P388、BEL-7402、Hela细胞的抑制率分别为27.40±4.71%,43.44±1.86%,30.20±1.16%,29.68±2.61%。镜检发现细胞形态都发生不同程度的变化。肿瘤细胞的生长抑制活性量效关系表明KP对P388的IC50为120μg/mL,对BEL-7402的IC50大于200μg/mL,细胞毒作用呈剂量和时间依赖性,对P388的抑制活性要高于对BEL-7402的抑制活性。抑菌实验也显示KP具有广谱抗微生物性能,对供试的2种革兰氏阳性菌和5种革兰氏阴性菌都有一定程度的抑制活性,量效关系均呈剂量依赖型,最小抑菌浓度(MIC)为10μg/mL～40μg/mL。另外,多酚对革兰氏阴性细菌中的大肠杆菌、绿脓杆菌抑制效果较明显,对真菌如青霉、白色念珠菌亦有抑制作用。4.海带多酚的分离纯化及其各部分活性检测:利用有机溶剂萃取分离、AB-8大孔树脂吸附分离、SephadexLH-20凝胶层析等技术对KP进行分离纯化,最后得到4个峰组分,分别为A1、A2、B1、B2,并筛选出了具有较高活性的多酚组分。各组分肿瘤抑制活性大小依次为:A2 > A1 > B2 > B1,浓度为70.42μg/mL时,组分A2对BEL-7402和P388的抑制率分别为61.96±7.02%,40.47±8.70%;各组分的抑菌活性大小依次为:A2 > B2 > A1 > B1,在酚浓度为70.42μg/mL、45.68μg/mL的条件下,对4种菌均有抑制效果且比粗提物和树脂分离产物同等浓度下抑菌圈直径大。5.海带多酚理化性质初步研究:对KP的物理化学性质如溶解性、折光率、特征显色反应、pH稳定性、热稳定性及光谱特征进行了初步研究。结果表明:KP易溶于中极性溶剂,而在非极性溶剂和极性溶剂中溶解性较差;在不同溶剂中的折光率大小依次为乙酸乙酯>乙醇>丙酮>甲醇>水>氯仿=乙醚;特征显色反应表明KP具有酚类物质的共性。另外,KP在pH2.0～11.0和40～100℃的范围内都有肿瘤抑制活性,具有良好的pH稳定性和热稳定性。组分A2与PG、间苯三酚的紫外吸收谱图具有相似之处。组分A1、A2、B2与PG、连苯三酚的红外谱图都具有明显的酚羟基伸缩振动;都有芳香环骨架伸缩振动;在指纹区都有酚的特征吸收峰,在官能团区也较为相似,初步推断KP具有PG或连苯三酚相近或相似的结构。6.海带多酚的抗肿瘤活性和抗菌活性机理初步探讨:以显微镜观察药物处理前后细胞的形态变化,考察了多酚对自由基、致癌物的清除能力;以流式细胞术分析了多酚对肿瘤细胞的凋亡率及对细胞增殖周期的影响,并测定了菌体系统的紫外吸收变化。结果表明:KP处理P388和BEL-7402细胞48h后,镜检细胞形态发生较大变化,与正常对照组差异显著;在100μg/mL的浓度下,KP具有一定的清除自由基和致癌物的活性,对·OH、O2·-、NO2-清除率分别为75.37±5.68 %、55.48±4.35%、50.51±6.46%。流式细胞术表明多酚对P388肿瘤细胞的凋亡率及细胞增殖周期存在影响,其中A2组分凋亡峰极为显著。加入多酚的菌悬液280nm处的吸光度均明显增高,且对革兰氏阴性菌更为明显。更多还原

【Abstract】 As a class of natural material, which is rich in various biological activities, plant polyphenol links closely with human life. It has effected on human digestion, nutrition, health and has been widespreadly used in the area of food, drug and cosmetic industry.In this paper, the extraction, isolation, anti-tumor activity and anti-bacterial activity of the polyphenols from fresh Laminaria japonica Aresch were studied systematically, and some characteristics, mechanism and application were also investigated. This work may provide primary data for further researching on the relationship between the structure and function of phlorotannins, as well as provide a new way for high-value utilization of kelp and exploiting some new anti-tumor medicine and food antiseptic.This study includes six parts of content as follows:1. Selection of measuement method of phlorotannins on exudation rate The kelp could be synthetically utilized to extract phlorotannins and other by-products. Based on comparison study on the two methods of determination phlorotannins, ferrous tartrate colorimetry and Folin-Denis method, ferrous tartrate colorimetry with PG as reference material was chosen because of its facility and credibility. Phlorotannins content of fresh kelp was slightly higher than dry kelp under the same extraction conditions.2. Determination of the optimal conditions for extraction of phlorotanninsAdopt fresh kelp as materials, ultrasonic and microwave extraction as pretreatment, extraction rate of phlorotannins as target, on the base of the single factor experiment and orthogonal test, the optimal extraction conditions of kelp polyphenols were researched. The results show that optimal extraction conditions of kelp polyphenols from Laminaria japonica Aresch are the ratio of sample weight to the solvent volume 1:7(g/mL), 85% ethanol, temperature 70℃, extraction times 2, extraction time 4h, and the exudation rate of kelp polyphenols is 2.08%.3. Investigation on anti-tumor activity and anti-bacterial activity of the crude extracts of brown algae phlorotanninsThe anti-tumor activity of crude extract of phlorotannins for human lung adenocarcinoma cell (A549), murine leukemic cell (P388), human hepatocellular carcinoma cell (BEL-7402) and human cervical cancer cell (Hela) had been determined respectively by MTT method in vitro. The results showed crude extracts has anti-tumor activity and the inhibitory rate were 27.40±4.71%, 43.44±1.86%, 30.20±1.16%, 29.68±2.61%, respectively at the concentration of 100μg/mL. The result of the microscope detection showed that the morphologic features of tumor cells were changed from the normal control group. Dosage-effect relationship showed the half-maximal inhibitory concentration (IC50) for P388 cell and BEL-7402 cell were 120μg/mL and >200μg/mL respectively. Moreover, the cytotoxic effect to P388 and BEL-7402 seems to be dose-dependent and time-dependent and the inhibitory rate for P388 was superior to that of BEL-7402.The anti-bacterial activity of crude extracts of phlorotannins had been determined by flat plate slotting to inhibit growth method in vitro, respectively. The results of bacteriostatic experiment showed that 2 species of gram-positive cocci and 5 species of gram-negative bacilli were inhibited by crude extract which had wide antimicrobial performance. Dosage-effect relationship was dose-dependent and the minimal inhibitory concentration (MIC) was 10μg/mL～40μg/mL. Meanwhile, the inhibitory effect of crude extract to Escherichia coli, Pseudomonsa aeruginosa was relatively obvious and fungi such as Penicillium, Candida albicans were also inhibited.4. Isolation and purification process of the extract crudesThe crude extracts could be divided into 4 kinds of bioactive fractions (A1, A2, B1, B2) after extraction with organic solvent, adsorption and desorption with AB-8 macroporous resin, column chromatography with sephadexLH-20. All bioactive fractions were compared in each purification process. Screening results of antitumor and antibacterial activity showed A2 > A1> B2 > B1 and A2 > B2 > A1 > B1, respectively. And the inhibitory rate of fraction A2 was 61.96±7.02% and 40.47±8.70% to BEL-7402 and P388 respectively at the concentration of 70.42μg/mL, and antibacterial activity was also increased compared to front parts.5. Primary research on the physical and chemical characteristics of phlorotanninsThe physical and chemic characteristics of phlorotannins such as dissolubility, light refraction, characteristic color reaction, pH stability, heat stability and spectral feature were determined primaryly. The results showed that the dissolubility of phlorotannins in CH3OH, CH3CH2OH, C2H5COOC2H5 were better than that in H2O, CH3Cl, C2H5OC2H5. Refractive index of phlorotannins in different solvents was C2H5COOC2H5 > CH3CH2OH > CH3COCH3 > CH3OH > H2O > CH3Cl = C2H5OC2H5. Characteristic color reaction showed kelp phlorotannins had the commonness of phenols. Otherwise, kelp phlorotannins had pH stability, thermal stability between pH 2.0～11.0 and 40℃～100℃. Ultraviolet absorption spectra of component A2 was similar to PG and phloroglucinol dehydrate. IR spectras of fraction A1, fraction A2 fraction B2, PG and pyrogallol had obvious expansion and vibration of phenol hydroxyl, aromatic rings. Kelp phlorotannins compound is preliminarily deduced as similar structure of PG or pyrogallol.6. Primary discussion of anti-tumor activity and anti-bacterial activity mechanism of phlorotanninsThe change of cell morphology was observed by fluorescent microscopy, and scavenging ability of free radicals and carcinogenic agents were discussed. At the same time, we analysed cell apoptosis, cell cycle by flow cytometry and detected UV absorption. The results showed that microscope detection the morphologic features of P388 and BEL-7402 tumor cells were changed from the normal control group after treated 48h. The scavenging rate of·OH, O2·-, NO2–were 75.37±5.68 %, 55.48±4.35%, 50.51±6.46%, respectively. The anti-tumor activity of phlorotannins was related with the cell apoptosis and cell proliferation cycle, especially obvious of A2 apoptosis peak, but the molecular mechanisms remain unclear. Ultraviolet absorption of the system containing phlorotannins and bacteria was increased at 280nm, especially for G-.更多还原