Study On The Extraction,Separation And Antioxidant Activity Of Anthocyanins From Lotus

Lotus belongs to the water lily family,which is a perennial aquatic herb.It is widely planted in China and rich in resources and one of the dual-purpose plants for medicinal and edible plants.The previous study have shown that the petals of lotus contain rich anthocyanins,which have the physiological functions of scavenging free radicals,anti-inflammation,anti-aging,inhibiting tumors and protecting eyesight,etc.,and have certain application value in food,medicine and cosmetics industries.However,lotus is mostly used as an ornamental plant,and its nutritional value is not fully utilized.The systematic study of anthocyanins in lotus and its biological activity can provide a better theoretical support for the utilization of lotus resources.In this study,anthocyanins were extracted from lotus.The optimum industrial process of extraction of lotus anthocyanins were determined by solvent method.The purification of lotus anthocyanins by AB-8 macro-porous resin was optimized.Lotus anthocyanins were separated by high speed countercurrent chromatography(HSCCC)and identified by liquid chromatography-mass spectrometry(LC-MS).The antioxidant activity of lotus anthocyanin was studied by in vitro antioxidant test and quantum chemical calculation.The main results were as follows:(1)The extraction conditions of lotus anthocyanins were optimized on the basis of single factor test using response surface methodology.With the help of software design-expert,the optimum extraction tech-nology were as follows:72.4%ethanol,extraction time 62.5 min,material-solbent tatio 1 g:26 mL.Anthocyanins content was 2.62±0.05 mg/1 g dry basis.(2)Through the static adsorption and desorption test of AB-8 macro-porous resin,the adsorption rate was 86.15%and the desorption rate was 85.84%.The purification process of anthocyanin from lotus flower by AB-8 macroporous adsorption resins was optimized.The results showed that the optimal dynamic adsorption conditions were loading of 2 g/L sample solution(pH 2)at a flow rate 1 mL/min,adsorption with 330 mL,and the optimal dynamic desorption conditions were that the desorption solution was 20%ethanol(pH 2)at a flow rate of 1 mL/min.Under these conditions,the purity of anthocyanin was increased from 4.32%to 23.7%.(3)The purified anthocyanin was separated by high-speed counter-current chromatography(HSCCC),and four components(Fr1,2,3,4)were obtained;Based on the scanning of UV-Visible wavelength,Fr2 and Fr4 were non anthocyanins,and Fr1 and Fr3 were anthocyanins with purity of 76.1%and 83.3%;Frl and Fr3 were identified by liquid chromatography-mass spectrometry(LC-MS).It was found that Fr1 contained one anthocyanin(Fr1-10.10),which was supposed to be malvacaine-3-O-glucoside and Fr3 contained two anthocyanins(Fr3-4.96,Fr3-6.77),which were supposed to be delphinidin-3-O-sambubioside,cyanidin-3-O-sambubioside.(4)Based on the in vitro antioxidant test of lotus anthocyanins,the results were as follows.The antioxidant activity of lotus anthocyanins was positively correlated with its concentration,and the antioxidant activity of Fr3 was higher than that of Fr1.In the experiment of scavenging ABTS·+,Frl and Fr3 showed strong scavenging ability,and the clearance rate of both anthocyanins was higher than 90%(92.63%,97.78%)at 50 ?g/mL.In the experiment of scavenging DPPH-,the scavenging ability of Frl and Fr3 is limited,and the free radical clearance rate was only 34.68%and 37.16%,lower than Vc.(5)The 3 anthocyanins(Fr3-4.96,Fr3-6.77,Fr1-10.10)were calcu-lated by software Gaussian 09W,and the results were as follows.The angles of A,C rings of three anthocyanins were-178.0158°,-178.2628°and-179.6753°respectively,and A,C rings had a good planar stucture.However,the included Angle of B C ring was greatly affected by C3 glycosyl substituents,and the included Angle of fr3-4.96,fr3-6.77 was 44.7617°,27.8501° and Fr1-10.10 was-174.3523°.Comparing the bond length of the same phenolic hydroxyl group with the atomic charge number of the hydroxyl group H,the order of antioxidant activity of 3 anthocyanins are as follows:Fr3-4.96>Fr3-6.77>Fr1-10.10,consistent with the results of antioxidant test.