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

Folium nelumbinis, the leaves of Nelumbo nucifera Gaertn, distributes in china extensively and are often considered as safe or harmless natural plant. Modern studies have demonstrated that ther e are various bioactive constituents in Folium nelumbinis, such as, Polyphenols, Flavonoids, Phenolic acids and alkaloids. These constit uents could exhibit excellent activity of scavenging free radicals, an ti-obesity, anti-tumor, bacteriostasis, anti-inflammatory and preventing cardiovascular disease. At present, this herb, as materials of functi onal foods, drugs or cosmetics is becoming more and more popular in china.At the same time, resources of Lotus leaves have not bee n utilized effectively and there are about only 1% Lotus leaves of effective use. It is possible to broaden the application range or enh ance the commercial value of Lotus leaf, if evaluation and searchin g of bioactive molecules in Lotus leaf was powerful advanced. This paper mainly covers four parts:Extraction of polyphenols from Lo tus leaf; Purification of crude polyphenols; Identification of polyphe nol components and antioxidant activity study. The main imvestigati ons and experimental results are listed as following:(1) The extraction conditions of polyphenols from Lotus le af were optimized on the basis of single factor test using response surface methodology., With the help of software design-expert, the o ptimal extraction parameters were 53% of ethanol solution concentra tion,103min of extraction time and 65℃of extraction temperature. Under these parameters, the yield of total polyphenols reached to 5 2.43mg GAE(gallic acid equivalent)/g dry lotus leaf powder and the result was conformable to the predictive result.(2) Combining static and dynamic tests, AB-8 Macroporou s resins were applied to the purification of crude polyphenols from Lotus leaf. Results indicated that the optimal adsorption conditions were as follows:the feeding concentration was 1.48mg/mL, feeding rate 2BV/h, and sample solution pH 5.0; the optimal desorption co nditons were 70% ethanol with a flow rate of 1BV/h. Under above conditions, the purity of polyphenols was increased from 29.18% t o 53.17%.(3) Different ethanol concentrations at a flow rate of 1BV/ h were used for desorption of polyphenols absorbed on Macroporou s resins and the desorption polyphenols were analyzed by HPLC-D AD methods. Results showed that the desorption rate of LP-30 was 59.63%, LP-50 was 21.20% and LP-70 was 4.87%. HPLC-DAD a nalysis showed that it was perfect for the seperation of polyphenol components of LP-30 and LP-50 at selected conditions. LP-30 had three peaks and LP-50 had seven peaks, all of which had similar UV-Absorbing characteristic compared with flavonoids.(4) The polyphenols of LP-30 and LP-50 were analyzed b y HPLC-ESI-MS methods. Two conponents were identified as Quer cetin-3-O-arabiopyranosyl-galactopyransoide, Quercetin-3-O-rhamnopyr anosyl-glucopyranoside and the third conponent was not identified f or LP-30. Seven conponents of LP-50 were identified as Isoquercitri n, Astragalin, Diosmetin-7-O-hexose,Isorhamnetin-3-Oarabinopyranosl-glucopyranoside,Isorhamnetin-3-O-rhamnose-glucoside,Isorhamnetin-3-O-glucoside,Malvidin-3-O-glucoside. Above all, the last one was fo und in Lotus leaf for the first time.(5) The antioxidant activity of polyphenols from Lotus leaf was measured. The total reducing power results indicated with the increase of total polyphenols concentration, reducing power increas es for the four samples and its activity order was LP-P> LP-30>L P-50>LP-C; In fenton system, the scavenging activity to hydroxyl radical was great for four samples and the IC50 of LP-30(0.059mg/ mL) was a little lower than LP-50(0.083mg/mL); All samples show ed stronger scavenging effect to nitrite in this experiment and the c learance rate of each sample exceed 97.00%; Lotus leaf polyphenols showed limited scavenging activity in hydrogen peroxide system. At the concentration of 0.200mg/mL, the clearance rate of LP-P an d LP-C were only 38.09% and 32.63% respectively, which were ab out half of positive control Vc(6) All calculations about three selected molecules were pe rformed with the Gaussian 03 software. The results indicated querce tin possessed most intramolecular hydrogen bonds and had a good planar stucture. The ring B, C of Isoquercitrin and Isorhamnetin-3-O-glucoside were torsional at different degrees, which was not favo r electron delocalization and thus weaken antioxidant activity; Comp aring the electron population of most active H atoms from phenolic hydroxyl groups in these three molecules, the order was as follow s:quercetin H18>Isoquercitrin H20> Isorhamnetin-3-Oglucoside H18, which was agree with results of reletive experiments; The antioxid ant activity of three molecules were predicted with the data of Hig hest occupied molecular orbital energy(EHOMO) or Energy gap(Egap), and its results were in agreement with the conclution of H atom el ectron population analysis.