Study On Extraction, Isolation,Purification And Antioxidant Activity Of Flvonoid In Lotus Leaf

Lotus leaf was a kind of plant proclaimed by National Ministry of Health, could be used both in medicine and food field. Lotus leaves were rich in many kinds of bioactive components, such as lotus leaf essential oil, alkaloid, polysaccharide and flavonoids. Usually the flavonoids from lotus leaf as its main active substance. As report, flavonoids have many biological activities, such as antioxidant, hypoglycemic, hypolipidemic and enhance immune activity. Through the study on extraction, separation, purification, structure identification, antioxidant and antimicrobial activity of flavonoids, this work attempt to select a group of natural antioxidant drug with potential pharmaceutical value. The main results of this study were as follows:1. Extraction and optimization of flavonoids from lotus leafCrude flavonoids were extracted from lotus leaves using distilled water. Respond surface methodology (RSM), based on Box-Behnken design was employed to obtain the best possible combination of extraction time, water to the raw material ratio and extraction temperature for maximum flavonoids extraction. The experimental data were fitted to a second-order polynomial equation using multiple regression analysis and also analyzed by appropriate statistical method (ANOVA). The best extraction conditions were as follows:extraction time96min, water to the raw material ratio of30, extraction temperature64℃, under these conditions, the crude flavonoid was3.86%, very close to the predicted value of3.74%.2. Separation and purification of flavonoids from lotus leafSemi-preparative HPLC method was developed to separate the flavonoids which has purified by AB-8macroporous resin. A highly efficient separation system was established to divide the flavonoids from lotus leaves. The best chromatographic analysis condition just as follow:the chromatographic column was Waters C18reverse phase column (150mm×10mm I.D.,5μm), the mobile phase A: acetonitrile, mobile phase B:ultra pure water (containing0.1%formic acid), the injection volume was600μL, the flow rate was3mL/min, column temperature was30℃,the detection wavelength was254nm, gradient elutionconditions were:0-20min5%A,95%B--40%A60%B.3. Structure determination of flavonoids from lotus leaf UV spectra with shift regents and LC-MS methods were used to determine the structure of the flavonoids. Three flavonoids were designated as quercetin-3-O-sambubioside, hyperin, quercetin-3-O-glucuronide, isoquercitrin and quercetin.4. In vitro antioxidant activity of flavonoids from lotus leafThe Fenton reaction method and pyrogallol autoxidation method were utilized to determination and evaluation the antioxidant effect of the three kinds of flavonoid (separated by semi-preparative HPLC). The experimental results showed that the flavonoids from lotus leaf have a strong ability to scavenge hydroxyl free radical and superoxide anion free radical.5. Calculation and analysis of flavonoid antioxidant structure-activity relationshipThis research adopts Gaussian03program, as DFT-B3LYP/6-311G (d) on flavonoid molecule to optimize the parameters of geometry, the molecular structure, electronic properties, and stability of free radicals and other aspects of the theoretical calculation. It has been observed from the harmonic vibration frequencies that all the conformations for these radicals have no imaginary frequency. Theoretical calculation could explain and illustrate the effect of structure on the antioxidant activity of flavonoid molecules from the theory angle. The theoretical calculations show that the flavonoid molecules of C-3glycoside substituent have a negative impact on the antioxidant activity, the more the number of glycosides, antioxidant activity is weak.6. Study on the antibacterial activity of flavonoids from lotus leafThe common contaminating bacteria in food were chosen to study the antibacterial activity of flavonoids. Bacteriostasis diameter and half inhibitory rate were used as tracking index to study the antibacterial properties and antibacterial mechanism of flavonoids from lotus leaf. Quercetin owns the highest antibacterial activity in lotus leaf flavone components. The inhibitory effect against gram positive bacteria was stronger than gram negative bacteria; weak inhibitory effect on yeast; almost no effect on the fungus. Inhibition effect on the test bacteria was:Staphylococcus aureus, Bacillus subtilize, Escherichia coli.