Study On The Isolation,Purification And Antioxidant Activity Of Polyphenols From Ficus Pumila Var.Awkeotsang Defatted Achenes

Jelly fig(Ficus pumila var. awkeotsang (Makino) Corner) is a woody vine belonging to Moraceae family. Water extract from jelly fig achenes has been utilized to produce jelly curd for making a popular drink in Taiwan. However the residual achenes after jelly curd making are discarded. For the purpose of sufficient utilization of these resources, the present study, jelly fig achenes cultivated in Minhou was used as experimental materials. Based on optimization of the extraction and purification conditions of polyphenol compounds from defatted achenes, the structures of polyphenol compounds were preliminary analyzed. Furthermore, systematic researches were carried out on its antioxidant activity and free radical scavenging capacity in vitro. The main results are as follows:1. GC-MS analysis demonstrated that, jelly fig seed oil was rich in unsaturated fatty acids including a-linolenic acid (52.67%), linolenic acid (20.81%) and oleinic acid (15.10%).The antioxidant activity of leaf and paroecious syconia of jelly fig were compared by using three evaluating systems, including FRAP method, DPPH· method and TEAC method. The result showed that the ethanol extract solution of the male flower in male syconia and achene in female syconia had higher antioxidant activities, and higher content of total phenolics. But from the perspective of synthetic utilization of resource, this work chooses the degelatinized achene of jelly fig to carry on further research.2. By using response surface analysis, the optimal conditions for the ultrasonic-assisted extraction of polyphenols from defatted achene were as follows: ethanol concentration,89%; solid to liquid ratio,22:1; temperature,30℃.The average validation extraction ratio of defatted achene polyphenols was3.21%, compared with the theoretical prediction value(3.25%), the relative error was1.34%.3. D101resin was selected from seven kinds of macroporous resins as a suitable material to separating the polyphenols from the jelly fig and the factors affecting the adsorption-desporption process were researched. The results showed the optimum adsorption conditions were as follows:intial extract solution concentration about2.3mg·mL-1, the adsorption time for about8hours, the pH value of the extract solution had no influence on the efficiency of adsorption; for desporption:elution solvent,70%ethanol, flow rate,1.0mL·min-1.Under the above optimal conditions, the purity of polyphenol extract was58.49±1.14%. After treated with D101resin, the mass fraction content of polyphenol in the extracts had increased16.87%than the defatted achene. The results showed that D101resin revealed a good ability to separate defatted achene polyphenols. Therefore, D101resin could be applied as an effective adsorbent for the production at large-scale.4. Polyphenols enriching extract, which prepared with macroporous resin method, were purified by Sephadex LH-20column chromatography technology. Eluate was then pooled into three major fractions including FracⅠ, FracⅡ and FracⅢ. Frac Ⅰ had the highest yield, accounted for62.41%of the purified product and the contents of polyphenol was highest, too. The results demonstrated Sephadex LH-20had effect on the isolation of polyphenol extract from jelly fig.5. The methanol solution of resin enrichment had max absorption peak at280nm. By comparing HPLC chromatograms of acid hydrolysate to the methanol solution of the polyphenolic resin extracts, the polyphenol extracts probably contained proanthocyanidins. The extracts of resin enrichment had the contents of total phenolics (465.67mg·g-1), flavanol (95.52mg·g-1), and proanthocyanidin (429.41mg·g-1). The proanthocyanidin accounted for73.41%of the total phenolics. The result prompted that proanthocyanidin was the main contributor to the antioxidant activity of the polyphenol from jelly fig.6. The antioxidant activity of polyphenols extracts from jelly fig defatted achene, resin enriching extract and three purified fractions (including Frac I, Frac II and FracIII).These fractions were systematically evaluated by using seven in vitro assays.Both the polyphenols extracts and its purified fractions exhibited strong reducing power, and higher than the positive control BHT. Resin enriching extract had the strongest reducing power with50%inhibition concentration (pIC50) of0.114mg·mL-1the value of FRAP was4.13FeSO4mmol·g-1.The statistic analysis showed that the50%inhibition concentrations and the value of FRAP both had certain associativity to their total phenolics of different extracts (R2>0.76). The reducing power of samples were as followed:resin enriching extract>FracⅠ>FracⅡ>raw extract>FracⅢ.The different polyphenol extracts had excellent scavenging activity on ABTS·+and DPPH·radicals. The scavenging rate increased with concentration increasing, and there was significantly linear correlation between scavenging rate and concentration with correlation coefficients (R2>0.97). According to the value of TEAC on ABTS·+, the ABTS·+scavenging activity of samples were as followed:resin enriching extract>FracⅡ>FracⅠ>raw extract>FracⅢ. The statistic analysis showed that the value of TEAC and its total phenolics had significantly positive correlation with correlation coefficients(R2) of0.9543. According to50%inhibition concentration (picso) on DPPH-and the DPPH-radical scavenging activity of samples were as followed:FracⅠ> FracⅡ>raw extract>resin enriching extract>FracⅢ. The result had some differences from the reducing power, the value of FRAP and TEAC.The different polyphenol extracts displayed strong scavenging abilities on-OH and O2-·, and the scavenging rate increased along with the increasing of concentration. In the range of tentative concentration, there was significantly linear correlation between scavenging rate and concentration with correlation coefficients (R2>0.99). Hydroxyl radical(·OH) scavenging activity of samples were as followed:resin enriching extract> FracⅡ> FracⅠ> FracⅢ> raw extract. Resin enriching extract had50%inhibition concentration (picso) of0.066mg·mL-1on·OH. According to50%inhibition concentration (picso) on·OH and the-OH radical scavenging ability of samples were as followed:FracⅡ>Frac Ⅰ>resin enriching extract>FracⅢ>raw extract.The different polyphenol extracts were showed certain inhibition of lipid peroxidation in β-carotene/linoleic acid systems. Resin enriching extract had the strongest inhibiting activity in all extracts and the inhibiting rate was62.09%at concentration (p) of1.00mg·mL-1.The lipid peroxidation-inhibiting activity of samples were as followed:resin enriching extract>Frac Ⅰ>FracⅡ>raw extract>FracⅢ.7. The different polyphenol extracts exhibited certain protecting activity against DNA strand scission by·OH on pUC18DNA, and stronger than the positive control Rutin. FracIII had exhibited the best protecting activity against DNA strand scission by oxidized damage. To sum up, the polyphenol from jelly fig belonged to procyanidin and had strong free radical scavenging capacity, antioxidant activity and exhibited certain protecting activity against DNA strand scission by oxidized damage. We could see that degelatinized and defatted achene from jelly fig had potential value of exploitation and utilization in the natural field of free radical scavenging capacity. However, further studies of chemical structure and mechanism of the antioxidant component are needed.