Study On Antimicrobial Components In Lotus Leaves

Lotus leaf is the department lotus of Nelumbo Nucifera Gaertn, which is abundant in China, mainly distributing at the Yangtse River Basin, Yellow River Basin and Zhujiang River Basin. Lotus leaf is the medically edible thing of dual purpose, for it contains many functional components of alkaloids, flavonoids, volatile oils, organic acid and steroids. Flavonoids, alkaloids and volatile oils are the main antibacterial components in lotus leaf. Extraction process, composition isolation and analysis, chemical structure identification, and antibacterial activities of the three components Were studied. Exploitations and prospects of natural lotus leaf bacteriostat were discussed.The main results were as follows:1,The extraction rates of crude extract, flavonoids, alkaloids and volatile oils from lotus leaves were high, and they had strong inhibition on bacteria activities but no distinct inhibition on yeast and mold. Among the extracts by solvents of different polarity, extracts by solvents ethanol, n-butyl alcohol, ethyl acetate had high antibacterial activities, illuminating that flavonoids and their aglycones, alkaloids and their salts were the main components with the antibacterial activities. The experiments also indicated that the extraction rate by solvent ethanol was the highest, and the antibacterial activity was the strongest. Plus, ethanol can be condensed quickly at low temperature, so it is the ideal solvent applied to extract natural antibacterial components from lotus leaves.2,Artificial Neural Network (ANN) combined with traditional orthogonal design, anewmethod of test data analyzing and processing was put forward, and a neural network mold was set up. By simulating, evaluating and optimizing, the optimal extraction conditions of flavonoids were obtained as follows: ratio of solid to liquid 1:30, extraction temperature 75℃, extraction time 1h, ethanol concentration 50%; the optimal extraction conditions of alkaloids: pH value 2.5, extraction temperature 85℃, extraction time 1.75h, ratio of solid to liquid 1:35. Antibacterial activities experiments were carried out, and the results showed: the extraction rates of flavonoids and alkaloids by optimal process were higher than general process, and antibacterial activities were stronger; the characteristics of flavonoids from lotus leaves were stable, and the antibacterial activities were slightly affected by pH value and temperature. The antibacterial activities of alkaloids from lotus leaves were slightly affected by temperature, but obviously affected by pH value. The antibacterial activities were much stronger in alkaline environment than in neutral or acid environment.3,Steam Distillation (SD), Solvent Extraction(SE) and Supercritical CO₂ Fluid Extraction (SFE-CO₂) were applied to extract volatile oils from lotus leaves. The extraction rates, process efficiency, sensory characteristics and antibacterial activities were compared and analyzed. The results showed that SFE-CO₂ was the ideal method to extract volatile oils from lotus leaves. GC-MS was applied to isolate and analyze the components of extracted volatile oils. 76 components were detected, among which SD and SE detected 23 components respectively, while SFE-CO₂ detected 52 components. The main ingredients were terpenes, alcohols, esters, ketones and alkyls, etc. Components of volatile oils extracted by the three methods varied, only 5 components in common.4,Thin Layer Chromatogram (TLC) was applied to isolate and identify alkaloids from lotus leaves, and analytical High Performance Liquid Chromatography (HPLC) was applied to analyze. The results showed: when TLC solvent system was: chloroform - methanol - diethyl amine = 90:9:1, alkaloids from lotus leaves were well isolated, and five components were isolated and identified by spraying Dragendorff reagent. The optimal conditions of HPLC solvent system were: methanol- water - diethyl amine = 75:25:0.0125. Under the conditions, components of alkaloids from lotus leaves were separated very well, and ideal RP-HPLC peaks were obtained.5,Methods of High Speed Counter Current Chromatography (HSCCC) and preparative High Performance Liquid Chromatography (PRE-HPLC) applied to identify and purify alkaloids from lotus leaves were set up. When solvent system of HSCCC was: chloroform - methanol - water (pH=4.00) = 4:3:2, speed of chromatogram was 700rpm, flow speed of the mobile phase was 2mL/min, four pure components relatively were attained by isolation and purification. When solvent system of PRE-HPLC was: acetonitrile - waterdiethyl amine = 55:45:0.0125 and acetonitrile - water- diethyl amine = 60:40:0.0125, four pure components relatively were attained. By identifying the obtained components by TLC, three alkaloids components were detected, among which one was nuciferine. The other two were N-Nornucirerine and O- Nornucirerine, identified by Nuclear Magnetic Resonance (NMR) and Mass Spectrum (MS).6,In order to exploit natural bacteriostat of lotus leaves, optimal extraction process and antibacterial characteristics were studied. Quadratic regression orthogonal design was applied, non-linear mathematic model was set up, non-linear optimization was applied, and the actual manufacturing situations were considered, the optimal extraction Condition of antibacterial of lotus leaves were obtained as follows: extraction temperature 50℃, ethanol concentration 67%, solvent times 40, extraction time 60min. Antibacterial experiments results showed that extractions of bacteriostat of lotus leaves had significant antibacterial activities, and the antibacterial characteristics were stable, slightly affected by environment Conditions, such as pH value, temperature, concentration of cations. Also, the extractions exhibited strong antibacterial activities very soon. Thus, it hasabright prospect in the exploitation and application to extract natural bacteriostat from lotus leaves.