Extraction, Purification, Structural Identification And Biological Functions Of Anthocyanins In Purple Cabbage

Anthocyanins extracted from purple cabbage with strong antioxidation can remove the free radicals inhuman body. The anthocyanins have gain much attention because of their biological functions such asvariation resistance, anti-tumor, anti-allergic and protecting the gastric mucosa. At present, the researchconcentrates on extraction, separation and purification methods, thermal stability, oxidation resistance andphysiological functions. In this thesis, extraction of anthocyanins from purple wild cabbage was optimized.The anthocyanins were preliminarily extracted and purified from fresh purple cabbage with macroporousresin. The structure and anti-free radical activities of the components of effluents from polyamide sheetschromatography were measured. The binding mechanism of cyanidin-3-O-glucoside to BSA was studied byspectroscopy. The interactions of cyaniding-3-O-glucoside with DNA were also investigated by UVabsorption spectroscopy.1. Anthocyanin from purple cabbage was extracted and purified using solvent extraction, extractionand thin-layer chromatography. The results showed that the optimum technological was extracting twice at25oC for1hours with3.5%hydrochloric acid methanol solution and solid-liquid ratio of30ml/g. In orderto determine the optimum macroporous resin and condition for purifying pigments extracted from thepurple cabbage, the static adsorption and desorption properties of five different resins were investigated.The result were as follows: the best chosen resin was D101, the optimal adsorption conditions were:temperature,25oC, adsorption time,75-90min, the optimal desorption conditions were: solvent70%ethanol, desorption time,15-30min, temperature,25oC.2. The activities of the purple cabbage anthocyanins separated and purified using polyamide thin-layerchromatography separation toward free radicals such as hydroxyl radical, superoxide anion radical havebeen measured, and the result indicated that crude anthocyanin products have the abilities of scavenginghydroxyl radical and superoxide anion radical. In the same concentration, the order of the inhibitory effecton the two free radicals is catechins> tea polyphenol> crude anthocyanin products. The four anthocyanincomponents spreading out on the polyamide thin-layer showed the inhibitory effect on the two free radicalsin varying degrees. With respect to inhibition ratio of hydroxyl radical, the average inhibiting ratio of theanthocyanin obtained is5.7times that of the catechins,2.8times that of Vc and equivalent to that of C3G.At the aspect of inhibitory action of superoxide anion radical, the average inhibiting ratio of theanthocyanin is3.9times of the values for catechins,4.8times of the values for Vc and inferior to C3G.3. Liquid chromatography-mass spectrometry (LC-MS) and Ultraviolet-visible spectrometry were jointly applied to determine the components and structures of purple cabbage anthocyanins. The resultsshowed that two aglucon and five anthocyanins were identified out, they were peonidin, cyanidin andcyanidin-3-glucoside, peonidin-3-sinapylsophoroside, sinapylpeonidin, ferulylpeonidin,cyanidin-3-sinapylsophoroside-5-glucoside. Both sinapylpeonidin and ferulylpeonidin have not yet beenreported.4. The interaction of pelargonidin with BSA was studied by fluorescence spectroscopy, UV absorptionspectroscopy and synchronous fluorescence spectroscopy. The results showed that Cy-3-O-Glu couldinduce an endogenous fluorescence quenching of BSA under a mechanism of static quenching. Thequenching rate constants at25oC,30oC and37oC were determined to be1.81×10~4，2.63×10~4,3.10×10~4L·mol－1, respectively; the binding constants4.21×10~4，5.92×10~4，8.887×10~4L·mol－1, respectively. Thenumber of binding site of the static quenching was calculated. The Cy-3-O-Glu had only a slight influenceon the BSA conformation by the fluorescence spectra. According to the theory of F rster nonradiativeenergy transfer the interacting distance of Cy-3-O-Glu and BSA was estimated to be3.29nm nm with anefficiency of0.0288. By analysis of thermodynamic parameters, the binding of Cy-3-O-Glu with BSA wasmainly attributed to the hydrophobic interaction. And the impact of the change of ionic strength or thepolarity to the interaction Cy-3-O-glu with bovine Serum Albumin was studied. The results show that, withthe increase of ionic strength or polarity, The Cy-3-O-glu to the intensity of the BSA fluorescencequenching weakened, but the chemiscal structure of the C3G-BSA compound does not change.