荔枝蜜的花源标识物及枣花蜜诱导HepG2细胞凋亡研究

发布时间：2018-04-23 07:05

本文选题：荔枝蜜 + 枣花蜜　；参考：《西北大学》2017年硕士论文

【摘要】：荔枝蜜和枣花蜜是我国市场上较为常见的两大单花种蜂蜜。枣花蜜多产自我国北方地区,而荔枝蜜多产自南方地区。虽蜂蜜采自不同的植物源,色泽口感各有特色,但都具有丰富的营养价值。然而,目前对荔枝蜜和枣花蜜的研究却有所不足,对花源标识物的研究以及在细胞水平对有害细胞的作用均鲜有报道。鉴别蜂蜜花源标识物是判别蜂蜜真假、区分掺假蜂蜜的一种重要手段。而细胞活性研究则对了解蜂蜜的药用价值以及功能开发具有重要意义。因此,本课题围绕荔枝蜜的花源标识物和枣花蜜对HepG2人肝癌细胞的促凋亡作用进行初步探究。1、构建了采自中国不同地区的13批荔枝蜜和13批枣花蜜的HPLC-DAD指纹图谱,获取相关系数矩阵数据,并对样品进行聚类分析。指纹图谱结果显示,荔枝蜜共有28个共有峰,枣花蜜共有32个共有峰。于荔枝蜜中发现2种化合物,枣花蜜中发现4种化合物可作为其花源标识物。聚类分析结果表明基于相关系数矩阵进行最远距离聚类可在一定程度上分析样品的地理源,为地理源差异研究提供支持。2、对荔枝蜜中发现的2种花源标识物(B1和B2)进行分离纯化和结构鉴定。采用XAD-2吸附树脂和半制备型液相色谱分离,分别得到两种花源标识物22.5 mg和4.7 mg,经Q/TOF-MS验证,其分子量均为264,是脱落酸及其同分异构体。3、对荔枝蜜和枣花蜜的体外抗氧化活性进行研究,分别测定了总酚含量、DPPH·清除能力和氧自由基清除能力(ORAC)。结果显示,枣花蜜的总酚含量高于荔枝蜜,均值为525.1±72.35 mg GAE/kg。枣花蜜DPPH·清除率(IC50)均值和ORAC均值为32.55±4.16 mg/mL和3.39±0.85 μmol TE/g,明显高于荔枝蜜,且与总酚含量表现出正相关。因此,选择枣花蜜为研究对象进行下一步研究。4、研究了枣花蜜对HepG2人肝癌细胞的促凋亡作用,并探究了其诱导凋亡的机制。结果显示,经枣花蜜处理的HepG2细胞活力会随着枣花蜜浓度的增加而下降。枣花蜜可以引发HepG2细胞凋亡,使其细胞核发生破裂,凋亡特征明显,且凋亡细胞比例与枣花蜜的浓度也表现出浓度正相关性。此外,枣花蜜会引起线粒体膜电位降低、增加Bax和Bad蛋白的表达量,降低Bcl-2和Bcl-xL蛋白的表达量。同时,抑癌基因p53的表达量也伴随处理浓度增大而增加,caspase-9和下游蛋白caspase-3表达上调,最终导致凋亡。可见,枣花蜜可能通过p53介导线粒体依赖途径诱导HepG2细胞凋亡。
[Abstract]:Litchi honey and jujube nectar are two common single-flower honey in Chinese market. Jujube nectar comes from the north of China, while litchi honey from the south. Honey from different plant sources, color and taste have their own characteristics, but have rich nutritional value. However, the researches on litchi honey and jujube nectar are not enough, and there are few reports on the floral markers and the effects on harmful cells at the cell level. The identification of honey flower source marks is an important means to distinguish the true and false honey and to distinguish the adulteration of honey. The study of cell activity is of great significance to understand the medicinal value and function development of honey. Therefore, the effects of flower marker of litchi honey and jujube nectar on apoptosis of HepG2 human hepatoma cells were studied in this paper. The HPLC-DAD fingerprints of 13 batches of litchi honey and 13 batches of jujube nectar collected from different regions of China were constructed. The correlation coefficient matrix data were obtained and cluster analysis was carried out on the samples. The fingerprint showed that there were 28 peaks in litchi honey and 32 peaks in jujube nectar. Two compounds were found in litchi honey and four compounds in jujube nectar. The results of cluster analysis show that the remote clustering based on the correlation coefficient matrix can analyze the geographical source of the sample to a certain extent. To provide support for the study of geographical source difference. 2. To isolate and purify and identify the structure of two floral markers (B _ 1 and B _ 2) found in litchi honey. Two flower markers, 22.5 mg and 4.7 mg, were obtained by XAD-2 adsorption resin and semi-preparation liquid chromatography, respectively, and were verified by Q/TOF-MS. Its molecular weight is 264, which is abscisic acid and its isomer. 3. The antioxidant activities of litchi honey and jujube nectar in vitro were studied. The scavenging capacity of DPPH and oxygen free radical were determined respectively. The results showed that the total phenol content of jujube nectar was higher than that of litchi honey, and the mean value was 525.1 卤72.35mg GAE / KG. The mean DPPH clearance rate (IC50) and ORAC of jujube nectar were 32.55 卤4.16 mg/mL and 3.39 卤0.85 渭 mol TEP 路g, which were significantly higher than that of litchi honey, and had a positive correlation with total phenol content. Therefore, the effect of jujube nectar on apoptosis of HepG2 human hepatoma cells was studied, and the mechanism of inducing apoptosis was explored. The results showed that the cell viability of HepG2 treated with jujube nectar decreased with the increase of jujube nectar concentration. Jujube nectar could induce the apoptosis of HepG2 cells, and the apoptosis characteristics were obvious. The proportion of apoptotic cells was positively correlated with the concentration of jujube nectar. In addition, jujube nectar could decrease mitochondrial membrane potential, increase the expression of Bax and Bad, and decrease the expression of Bcl-2 and Bcl-xL. At the same time, the expression of tumor suppressor gene p53 was increased with the increase of treatment concentration, and the expression of caspase-9 and downstream protein caspase-3 was up-regulated, which resulted in apoptosis. Therefore, jujube nectar may induce apoptosis of HepG2 cells through mitochondrial dependent pathway mediated by p53.
【学位授予单位】：西北大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S896.1;TS201.2

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