樟树叶中木脂素提取分离及其生物活性研究

发布时间：2018-11-11 12:29

【摘要】：樟树主要产于我国长江以南地区。樟树枝繁叶茂,树形优美,不仅能吸收硫、氟等有害气体,还具有防尘、驱虫的功效,因此樟树常被用来美化环境、净化空气。樟树叶、枝具有散寒、止痛、补火、助阳的功效,除此之外还具有治疗各种痛症的功效。樟树叶中含有精油、多酚、黄酮、木脂素等天然活性物质。目前对樟叶中的精油、多酚、黄酮类物质研究的较多,但是对樟树叶中木脂素类物质的研究较少,本文是以樟树叶为原料,优化不同提取技术优化樟树叶中木脂素工艺条件,对粗提取物进行分离纯化并利用高效液相法(HPLC)分析分离物质的纯度,利用对DPPH自由基、羟基自由基、超氧离子自由基的清除效果和还原力的测试判断木脂素的抗氧化;通过对大肠杆菌、枯草芽孢杆菌、金黄色葡萄球菌、绿脓杆菌、沙门氏菌、黑曲霉、青霉的抑菌活性判断木脂素的抑菌效果;以宫颈癌细胞(HeLa)为研究对象,通过MTS,FCM,电化学法考察木脂素的抗癌效果。结果如下:(1)利用传统方法—乙醇回流法为基础方法,它主要是考察单因素乙醇浓度、料液比、回流时间对樟树叶中木脂素得率的影响,在单因素和正交试验的基础上优化提取工艺,最终得到最佳的工艺水平。最佳水平为:乙醇浓度为100%,料液比为1:4(g/mL),回流时间为2 h。(2)利用微波辅助技术提取樟树叶中木脂素类物质。主要是通过单因素试验和响应面试验对微波辅助提取工艺进行优化。得出响应面优化的最佳工艺条件为:微波时间5 min、料液比1:26(g/mL)、乙醇浓度80%、微波温度60℃。(3)利用超声波辅助技术提取樟树叶木脂素,结合单因素和响应面试验优化提取工艺,结果表明在料液比1:22 g/mL、超声时间3 min、乙醇浓度60%、提取时间78 min条件下,木脂素得率最大。(4)利用对DPPH自由基、羟基自由基、超氧离子自由基的清除效果和还原力的测试判断粗木脂素类物质的抗氧化性能。结果表明:木脂素具有较好的抗氧化活性,在此基础上对粗木脂素进行分离纯化。(5)通过大孔树脂、硅胶、LH-20、ODS等柱色谱,薄层色谱(TLC)对樟树叶中木脂素类物质进行纯化。分别利用紫外分光光度法(UV)、傅里叶红外色谱(FT-IR)、质谱(MS)、核磁共振(NMR)中的氢谱(1H-NMR),碳谱(13C-NMR)对其进行结构鉴定。根据紫外区的最大吸收峰,红外色谱不同波段的透射峰,质谱数据,核磁共振中的氢谱及碳谱中的吸收值最终确定樟树叶中的3种木脂素,分别为松脂素-β-D-吡喃葡萄糖苷、松脂素、芝麻素。通过外标法采用高效液相法(HPLC)对分离出的3种物质进行纯度鉴定。(6)本章则通过肉汤稀释法和纸片法测定从樟树叶中分离到的松脂素对枯草芽孢杆菌和绿脓杆菌的抑制作用。在此基础上利用光学显微镜(LM)、扫描电镜(SEM)、生长曲线、细胞膜的通透性说明了松脂素抑制枯草芽孢杆菌和绿脓杆菌的机制。(7)利用MTS法检测了不同浓度的松脂素对宫颈癌细胞(HeLa)和人正常上皮细胞(HUVEC)的抑制作用,发现松脂素以浓度-效应的关系抑制HeLa细胞和HUVEC细胞的生长,并且对HeLa细胞的IC50为200 μg/mL。利用流式细胞术(FCM)和Hoechst 33258染色技术检测了松脂素处理前后的HeLa细胞的凋亡情况和周期情况。(8)利用羧基化的碳纳米管和金纳米粒子溶液对玻碳电极进行修饰从而放大电化学信号。通过循环伏安法考察不同体积比羧基化的碳纳米管和金纳米粒子溶液对电化学信号的影响进行优化,最终确定了碳纳米管的体积与金纳米粒子溶液的比例。利用循环伏安法(CV)和交流阻抗谱(EIS)分别对不同细胞浓度和不同松脂素浓度对宫颈癌细胞进行测定。与传统方法相比较,电化学法快速简便的测定松脂素对宫颈癌细胞的诱导凋亡作用,为快速测定宫颈癌提供了一个潜在的方法。
[Abstract]:The camphor tree is mainly located in the south of the Yangtze River. The camphor tree is luxuriant, the tree is beautiful, not only can absorb the harmful gas such as sulfur, fluorine and the like, but also has the functions of dust-proof and insect-expelling, so that the camphor tree is often used for beautifying the environment and purifying the air. The camphor tree leaves and branches have the effects of dispersing cold, relieving pain, tonifying fire, and promoting yang, and has the effects of treating various pain diseases. The camphor tree leaf contains natural active substances such as essential oil, polyphenolic, flavone, and lignan. At present, the research of essential oil, polyphenols and flavonoids in the leaves of Cinnamomum camphora has been studied, but the study of the lignan in the leaves of the camphor tree is less, this paper is to optimize the process conditions of the lignan in the leaves of the camphor tree by using the leaves of the camphor tree as the raw material and optimizing the different extraction techniques. the crude extract is separated and purified and the purity of the separated substance is analyzed by high-performance liquid-phase method (HPLC), The antibacterial activity of Staphylococcus aureus, Pseudomonas aeruginosa, Salmonella, Aspergillus niger and Paecilomyces was determined, and the anti-cancer effect of the lignan was studied by MTS, FCM and electrochemical method. The results are as follows: (1) The method is based on the traditional method of ethanol reflux, which is mainly used to study the effect of single factor ethanol concentration, liquid ratio and reflux time on the yield of the lignan in the leaves of the camphor tree, and the extraction process is optimized on the basis of the single factor and the orthogonal test. and finally the optimal process level is obtained. The best level is that the ethanol concentration is 100%, the ratio of the liquid to liquid is 1: 4 (g/ mL), and the reflux time is 2 h. (2) the lignan in the leaves of the camphor tree is extracted by the microwave-assisted technique. The microwave-assisted extraction process was optimized by single factor test and response surface test. The optimum conditions for the response surface optimization were: microwave time 5 min, material liquid ratio 1: 26 (g/ mL), ethanol concentration of 80%, and microwave temperature of 60 鈩，

本文编号：2324835