Study On Analysis, Separation And Purification Of Alkaloids From Nelumbo Nucifera Leaves

Nelumbo nucifera GAERTN (commonly known as lotus), one of the most well known ornamental and economic perennial aquatic plants and now widely cultivated and consumed all over the world. Nelumbo nucifera leaves is a kind of naturals plant material used both in food and medicine field, involving nutrients and many biological activities. In order to further the study of biological activities of these aporphine alkaloids and to control the quality of this traditional Chinese cash crops and their products, better analysis and purification methods are urgently needed.The main results are indicated as follows:1. A novel method for the analysis of alkaloids in Nelumbo nucifera leaves was established by SPE-RRLC-Q-TOF. The crude sample was extracted by 1% HCl with ultrasound-assisted extraction, then purified by SPE column, and eluted with ammonia–methanol (5:95, V/V). After concentration, the residue was dissolved by methanol solution. The real sample was analyzed by RRLC-Q-TOF. A Welch Materials C18 column was applied in the RRLC separation using acetonitrile and water as mobile phase. The elutens were detected by Q-TOF to obtain the MS spectra with extract molecular weights under positive ion mode. Nine alkaloids were identified. This method can be used to rapidly determine the alkaloids of Nelumbo nucifera leaves.2. Three methods of high-speed counter-current chromatography(HSCCC) were successfully applied to the separation of alkaloids from crude extract of Nelumbo nucifera leaves. The conventional HSCCC separations were performed with a two-phase solvent system composed of tetrachloromethane–CHCl3–methanol–0.1MHCl at a volume ratio of (1/3/3/2, V/V). Two pH-Zone-refining CCC were performed with a two-phase solvent system composed of petroleumether–ethyl acetate–methanol–water (5/5/2/8, V/V) and MtBE-methanol-water (4/1/5, V/V), where triethylamine (10mM) was added to the upper organic stationary phase as a retainer and hydrochloric acid (5 mM) to the aqueous mobile phase as an eluent, respectively. The targets were obtained in a single run each with a purity of over 98% as determined by HPLC. The structures of the isolated compounds were identified by ESI-MS, 1H-NMR and 13C-NMR. pH-Zone-refining CCC was the best method in sample-loading capacity, high purity, and high concentration of the collected fractions. The present method may be applied to purification of various other alkaloids from natural products.3. Using ultrasonic extraction, O-nornuciferin, N-nornuciferin, nuciferine and roemerine in Nelumbo nucifera leaves were extracted and determined by the established HPLC method. This work provided a new method for the rapid quality control of Nelumbo nucifera leaves and its products. Four alkaloids were separated on a reversed-phase Welch Materials C18 column with a gradient elution using acetonitrile-1% triethylamine solution as mobile phase. Linear regression of O-nornuciferin, N-nornuciferin, nuciferine and roemerin in the concentration range of 0.1~50μg/mL(R > 0.999) was satisfied and the detection limit were 1.87 ng, 2.22 ng, 2.34 ng and 1.88 ng (S/N = 3), respectively. The method had good precision, recovery and can be used for the determination of alkaloids from Nelumbo nucifera leaves.