龙眼核多酚的分离纯化及其对小鼠烫伤愈合的影响

发布时间：2018-10-24 22:06

【摘要】：本文以龙眼加工废弃物龙眼核为研究对象,对龙眼核主要化学成分进行了测定;通过不同极性溶剂萃取、大孔树脂结合Sephadex LH-20凝胶对龙眼核多酚进行分离纯化,采用高效液相色谱(HPLC)和高效液相-电喷雾质谱(HPLC-ESI-MS)初步分析了龙眼核多酚的主要组成成分;在此基础上对龙眼核多酚各级组分进行了抗氧化、抑菌活性的初步研究,同时探究了龙眼核多酚对小鼠烫伤愈合的影响,主要结果如下:(1)主要化学成分分析结果表明,龙眼核中的淀粉含量最高,其次是蛋白质和还原糖,粗纤维、脂肪含量相对较低,且含有较多的多酚类物质,故可作为一种良好的营养源。(2)液-液萃取法分离龙眼核多酚的研究结果表明,乙酸乙酯萃取物的多酚含量最高,达到693.51±9.52 mg/g;在不同自由基体系中,4种萃取物均对?O2-的清除能力最弱,但对ABTS?+、DPPH?、?OH均表现出较强的清除效果,其中乙酸乙酯萃取物的抗氧化活性最强;除水层剩余物对沙门氏菌和大肠杆菌均无明显抑制作用外,4种萃取物对沙门氏菌、大肠杆菌、金黄色葡萄球菌均具有不同强度的抑制作用,其中乙酸乙酯萃取物的抑菌活性最为突出。(3)通过静态试验比较了7种大孔吸附树脂对龙眼核多酚的吸附及解吸效果,筛选出AB-8为最佳树脂,其吸附率和解析率分别为69.65%和94.47%。龙眼核多酚的最佳纯化工艺条件为:p H值3.5,上样浓度2 mg/m L,上样流速2 m L/min,洗脱剂70%乙醇,洗脱流速2 m L/min。在此条件下,龙眼核多酚的回收率为75.65%,产品得率为4.93%,纯度为65.84%。(4)采用不同浓度的乙醇溶液对AB-8树脂上吸附的龙眼核多酚进行梯度洗脱,发现各洗脱部分的多酚组成和含量存在一定差异,表明AB-8树脂可用于龙眼核粗多酚的初步分离;体外抗氧化、抑菌实验结果表明,相同多酚浓度下,30%乙醇洗脱物的抗氧化和抑菌活性总体上均优于其他洗脱部分。Sephadex LH-20凝胶对30%乙醇洗脱物的分离效果较好,能获得5个组分,经HPLC和HPLC-ESI-MS初步分析表明,龙眼核多酚的主要活性成分为柯里拉京、鞣花酸等化合物。(5)通过创面愈合率、生化指标测定及组织病理学分析来评价创面的愈合质量,结果表明:中、高剂量组在烫伤愈合前期对创面收缩有一定的促进作用,之后不同时间点的愈合率与生理盐水组相比无明显差异;一定剂量的龙眼核多酚溶液对创面组织和血清中蛋白质和羟脯氨酸的含量变化有一定影响;在炎性细胞浸润、成纤维细胞和上皮细胞增生、毛细血管生成、肉芽组织形成等方面对组织病理学进行评价,龙眼核多酚各剂量组与生理盐水组比较均无明显差异。因此,龙眼核多酚对小鼠深Ⅱ度烫伤愈合有一定的影响,但无明显促进作用。
[Abstract]:In this paper, the main chemical constituents of longan nucleus were determined, and the polyphenols in longan nucleus were separated and purified by macroporous resin and Sephadex LH-20 gel with different polarity solvent extraction. High performance liquid chromatography (HPLC) and high performance liquid phase electrospray ionization mass spectrometry (HPLC-ESI-MS) were used to analyze the main components of polyphenols in longan nucleus. The main results were as follows: (1) the main chemical component analysis showed that the starch content in longan nucleus was the highest, followed by protein and reducing sugar, crude fiber and fat content was relatively low. It can be used as a good nutrient source. (2) the results of separation of polyphenols from longan nucleus by liquid-liquid extraction showed that the content of polyphenols in ethyl acetate extract was the highest. In different free radical systems, the four extracts had the weakest scavenging ability to? O2-, but they showed strong scavenging effects on ABTS? and DPPH?,?OH, among which ethyl acetate extract had the strongest antioxidant activity. Except that the water layer residue had no obvious inhibition on Salmonella and Escherichia coli, the four extracts had different inhibitory effects on Salmonella, Escherichia coli and Staphylococcus aureus. The antibacterial activity of ethyl acetate extract was the most prominent. (3) the adsorption and desorption of polyphenols in longan nucleus by seven macroporous adsorption resins were compared by static test. The best resin was AB-8, the adsorption rate and resolution rate were 69.65% and 94.47% respectively. The optimum purification conditions of polyphenols in longan nucleus were as follows: p H value 3.5, sample concentration 2 mg/m / L, sample flow rate 2 mL / min, eluant 70% ethanol, elution rate 2 mL / min. Under these conditions, the recovery of polyphenols in longan nucleus was 75.65, the yield of the product was 4.93 and the purity was 65.84. (4) gradient elution of longan polyphenols adsorbed on AB-8 resin was carried out with different concentration of ethanol solution. It was found that there were some differences in the composition and content of polyphenols among the eluting parts, which indicated that AB-8 resin could be used for the primary separation of crude polyphenols in longan nucleus, and the results of antioxidation and bacteriostatic experiments in vitro showed that, At the same concentration of polyphenols, the antioxidant and bacteriostatic activities of 30% ethanol elution were better than those of other elution parts. Sephadex LH-20 gel had a better separation effect on 30% ethanol elution, and five components could be obtained. The results of HPLC and HPLC-ESI-MS analysis showed that: 1. The main active components of polyphenols in longan nucleus are Corilagin, tannic acid, etc. (5) the wound healing quality is evaluated through wound healing rate, biochemical index measurement and histopathological analysis. In the high dose group, the wound contraction was promoted at the early stage of scald healing, but the healing rate at different time points was not significantly different from that in the saline group. The changes of protein and hydroxyproline contents in wound tissue and serum were affected by a certain dose of longan nucleus polyphenol solution, and in inflammatory cell infiltration, fibroblast and epithelial cell proliferation, capillary angiogenesis, The histopathology was evaluated in granulation tissue formation and so on. There was no significant difference between each dosage group and normal saline group in the evaluation of histopathology of longan nucleus polyphenols. Therefore, polyphenols in longan nucleus have a certain effect on the healing of deep second degree scald in mice, but have no obvious promoting effect.
【学位授予单位】：华南农业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R151;TS209

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