Purification And Antitumor-activities Of Crude Polyphonel Extract From Sargassum Fusiforme

Polyphenols have a variety of biological activities including antioxidation, antitumor, antibacterial, antiviral, anticoagulant etc., and drawed much attection all over the world. One important natural source of polyphenols is brown algae, in which the Sargassum fusiforme stands for a high polyphenol bearing species, as many articles have reported. This research used Sargassum fusiforme as raw material, the solid to liquid ratio, extracting time, temperature and the ethanol concentration were studied to establish an optimal method for polyphenols’ extraction through single factor and orthogonal experiments. The crude extract of polyphenols was obtained upon the optimal extracting method. The following separation and purification was carried out through 3 protocols, employing fractional extraction, combination of extracting and macroporous resin, and sevag method, respectively. Through the above protocols, 7 fractions were obtained and subjected to HPLC analysis, and were further examined for in vitro anti-tumor activities against HepG2, RAW264.7, HT29, and A-549 cell lines. The results were as follows.1. The optimal polyphenol extracting parameters: 45% ethanol concentration, a 1:30 ratio of material to solvent, a 7h extraction time at 70 ℃, in which condition the total phenol yield was 4.85±0.06mgGA/g, and polyphenols’ concentration was 1.5% in the freeze-dried crude extract.2. Through the 3 separation protocols the crude extract was further purified. A. Using ethyl acetate and n-butanol, 3 fractions named E1(ethyl acetate extract), E2(n-butanol extract) and E3(water phase solute) were obtained. B. Using combination of extracting and macroporous resin ADS – 21, the optimal purification condition was: 1ml/min flow rate for obsorbing at pH 4.5, 0.3 BV of samples volume, using 70% ethanol as eluent at 1ml/min and ended at 2.6 BV of elution volume. Two fractions were eluted and collected named R1 and R2. C. Using sevag method followed by ethanol precipitation, 2 fractions—— S1(0-70% ethanol precipitated) and S2(70-90% ethanol precipitated) were collected. The polyphenols’ concentrations of the above 7 fractions were 8.8%、6.1%、3.8%、6.9%、4.0%、4.6%、5.3%, respectively. HPLC was employed to analyse the 7 fractions, and we found that a HILIC system was suitable for examining these fractions except for E1.3. The 7 fractions showed significant differences in anti-tumor activities. At a concentration of 0.8mg/mL, the crude extract only inhibited the viability of RAW264.7, while E1 showed remarkable inhibitive activities against all the 4 cell lines with inhibition rates all above 80%. E2 also showed inhibitive activities against HepG2 and RAW 264.7, with inhibition rates above 80%. For the water-dissolved fractions E3、R1、R2、S1 and S2, no significant effects on tumor cell viability were detected. However exceptionally, S1 showed inhibitive effect on RAW264.7.This study has provided theoretical foundation for developing polyphenols products using Sargassum fusiforme, and built a feasible technical route for anti-tumor compounds’ purification and localization. And also, the research provided significant information for separating and bioactive study of the polar compounds such as phenols in brown algae species.