Studies On Extraction, Purification And Activity Of Hesperidin From Gannan Navel Orange Peel

According to recent research, there are about 60 different citrus flavonoids monomers, including flavone, flavonol, flavanone and cyanin, et al. The most rich flavonoids in the citrus is hesperidin, which displays important biological activity. This papar selected the characteristic local resources in Jiangxi province-Gannan navel orange peel as raw material, the extraction and purification of hesperidin were studied, then the interaciton feature between hesperidin with DNA(IgG) was studied too.The results were as follows:1. On the basis of UV-vis, compared with different determination methods(direct determination method, NaNO2-Al(NO3)3-NaOH method, AlCl3 method and 75% ethanol containing 0.1% NaOH method), selected 75% ethanol containing 0.1% NaOH method as the measuration method to determine hesperidin in navel orange peel. At the same time, the purity quotient of hesperidin in the crude product was determined, which the result of UV-vis method(67.25%) was higher than HPLC(61.42%). To save time and cost, ultimately determined that UV-vis method was used in the process of extraction and purification, and HPLC was used to determine the final purity of the sample because of its higher accuracy.2. Basing on the experiment of single factor, the optimum extracting conditions of flavonoids from navel orange peel were obtained by the response surface methodology(RSM), the results were as follows:The optimum extracting conditions of water bath method:Temperature of 69.96℃, ethanol concentration of 50.46%(v/v), the ratio of liquid to solid of 25.91 mL/g and extracting time of 3.04 h were found to be the optimal conditions to achieve the highest yield of flavonoids (2.97%).The optimum extracting conditions of ultrasonic-assisted extraction:The temperature of 75℃, ethanol concentration of 58.91%, liquid-to-solid of 30.68ml/g, extracting time of 20.79 min were found to be the optimal conditions to achieve the highest value of degree of flavonoids extraction (3.298%). The practical yield rate of flavonoids was 3.351%.As can be seen by comparing, ultrasonic-assisted extraction method has more advantages than water bath extraction method. In this experiment, we used ultrasonic-assisted extraction method.3. By comparing static adsorption and desorption properties of the four macroporous resins including D101, AB-8, NKA-Ⅱand NKA-9, the result indicated that the D101 type macroporous resin is the best for separating and purifying hesperidin from navel orange peel. The optimum conditions are:injecting concentration 0.2 mg/mL, pH=5, injecting velocity 1.0 mL/min,60% alcohol as desorption solvent, desorption velocity of flow 0.5 mL/min and elution volume 3 BV. The analytical result show that the highest content of hesperidin after purification with D101 type resin is 94.82% by high performance liquid chromatography (HPLC). The enrichment factor of hesperidin is 1.54 with recovery rate of 70.27%. Thus, it is feasible to separate and purify hesperidin by D101 type macroporous resin.4. The interaction between hesperidin and IgG was studied by fluorescence spectroscopy and UV-vis in physiological buffer solution(pH=7.40). It was observed that there was a endogenous fluorescence quenching reaction of hesperidin to IgG. The quenching constants of hesperidin with IgG were measured at different temperature, and the quenching mechanism was showed as combined quenching. The thermodynamic parameters indicated that the interaction between hesperidin and IgG was driven mainly by hydrophobic force. The results of synchronous and 3D fluorescence showed that binding of hesperidin to IgG induced conformational changes of IgG.5. The interaction between hesperidin and ct-DNA was investigated by UV-vis, fluorescence spectroscopy, viscosity measurements and DNA melting techniques in physiological buffer(pH=7.40). The results suggested that hesperidin can bind to DNA, and the major binding mode is intercalation binding. The quenching mechanism was showed as static quenching. The binding constants of heperidin with DNA-EB at 298K,304K and 310K were calculated as 0.69×104,1.29×104 and 1.79×104, and the binding sites were all about 1.