Studies On Extraction, Isolation, Purification And Antioxidation Activity Of Flavonoids From The Fruits Of Avicennia Marina

As a main kind of mangrove, Avicennia marina mainly distributed in Guangdong, Guangxi, Fujian, Hainan, Taiwan, Hong Kong, Macao and other provincial regions, its forest area is more than 7000 hm². Annual production of Avicennia marina fruit up to 20 thousands tons in china, of which about 9000 tons one year in Zhanjiang, the fruit of Avicennia marina is rich in resources, but has yet to rational development and utilization. The studies of Avicennia marina focused mainly on bioactive substances of branches, leaves and roots, rarely on fruits. However, our previous studies found that Avicennia marina fruit is rich in flavonoids, worth to be developed and utilized. The technical parameters of extraction, isolation and purification of flavonoids from the fruits of Avicennia Marina collected from Zhanjiang was studied, and their scavenging activities were measured by the methods of DPPH, Fenton, pyrogallol autoxidation methods respectively in this paper, the theoretical basis on conservation and comprehensive utilization of the fruits of Avicennia Marina is hope to provide. The main research contents and conclusions were listed as followed:1,The dry fruits of Avicennia Marina was extracted with seven different solvent dipping at normal temperature to get methanol extract, water extract, 95% ethanol extract, acetone extract, n-butanol extract, ethyl acetate extract and petroleum ether extract. The results showed higher extraction rate and total flavonoids yield in methanol extract, water extract and 95% ethanol extract. Based on an overall consideration of various factors, ethanol-water was selected as the extraction solvent. Then Response Surface Methodology (RSM) was used to optimize the extraction technology of flavonoids from the fruits of Avicennia Marina. On the basis of single factor tests, a Central Composite Design (CCD) experiment was conducted and the response surface analysis was employed to investigate the influence of four main factors including temperature, extracting time, concentration of ethanol, ratio of solvent to raw material on the yield of flavonoids,and then a regression model was established. The results showed that the optimum extraction conditions were temperature 55℃, time 3.5h, concentration of ethanol 66%(v/v) and ratio of solvent to raw material 22mL/g. The predicted yield of flavonoids with the optimized procedure was 8.651%, while the practical yield of flavonoids was 8.613%, indicating a good fitness of the regression model and the actual process. In the above conditions, the weight percent of flavonoids in crude flavonoids of Avicennia Marina fruits(AFC) is 17.39%.2,The adsorption and desorption ability of 10 macroporous resins including AB-8,D101,S-8,ADS-17,FL-1,FL-2,FL-3,XAD16,NKA and X-5 were comparative studied, and AB-8 was selected for separation of the extracts from Avicennia Marina fruits. Then its dynamic absorbing behavior was studied, acrooding to the results of static and dynamic analyses on the efficiency for separation of flavonoids, the optimum conditions of column-separating method were determined as followings: 1.6cm (diameter)×40 cm (length), diameter height ratio of filler 1:22.5, column volume72mL, water as solvent of adsorbates and its pH 4.65, 80% ethanol as eluent and its pH 6.51, sample concentration 3mg/mL and current velocity of sample 1.5mL/min, loading amount 7.5 bed volume, eluting velocity 2mL/min and eluting reagent volume 2 bed volume. In the above conditions, the weight percent of flavonoids in the purified flavonoids from the fruits of Avicennia marina (AFP)is 88.67% and the purification multiples of 5.1. The AFP was further isolated into five components F₁, F₂, F₃, F₄, and F₅ by Sephadex LH-20 column chromatography, then qualitative analysis was made on the five components by ultraviolet spectrum, color reaction and HPLC method. The results showed that F₁, F₂, F₃ and F₄ are flavonoids while F5 which the hydrogen of C3-OH replaced is flavonols, and every component was not a single compound, but a kind of flavonoids with similar structure.3,Using clearance rate as index, scavenging activities on superoxide anion radicals(·O₂-), hydroxyl radicals(·OH) and DPPH·of the seven extracts, the AFC, the AFP and component F₁, F₂, F₃, F₄, F₅ was studied in this paper. The results showed that the antioxidant and radical scavenging activities of six extracts except petroleum ether extract on DPPH·were higher than on·O₂^-,·OH, while the scavenging activity on·OH was stronger than on·O₂^- of the extract of weaker polarity solvent acetone, n-butanol, ethyl acetate, and petroleum ether. The total antioxidant capacity of the extract by the strongly polar solvent water, methanol and 95% ethanol is obviously higher than that of the other solvent extract. The scavenging activity on three kinds of free radical of AFP is higher than that of AFC, and F₅ had the lowest scavenging activity on three kinds of free radical. The clearance rate on DPPH·and·O₂^- of AFC, AFP and five components were below Vc and rutin. The clearance rate on·OH of F₄, AFP and F₁ were greater than those of Vc, F₃, rutin, F₂, AFC and F5. The total antioxidant capacity had little difference of AFC, AFP and five components, between the Vc and rutin.