Study On Isolation, Identification, Antimicrobial Activity Of Alkaloids From Lotus Leaf

Lotus leaf is a kind of natural plant material used both in food and medicine field. In modern pharmacological studies, scientists had found that alkaloids of lotus leaf were known as the main type of bioactive constituents, which have the functions of reducing body weight, antibacterium, antioxidation, antivirus, et al. In this paper, supercritical CO2 fluid extraction and ultrasonic wave assisted extraction technology were used to extract the total alkaloids from lotus leaves. The extraction conditions were optimized based on orthogonal experiment and the response surface analysis method, respectively. Four kinds of alkaloids monomers of lotus leaf were separated and prepared through the high-speed countercurrent chromatography (HSCCC) and preparation liquid (Pre-HPLC). Furthermore, their structures were identified using UV and LC/MS. This paper also evaluated the antibacterial activities of total alkaloids and four monomers, and the structure-activity relationship was preliminarily analyzed. The main conclusions were as follows:(1) The optimum extraction conditions of supercritical CO2 fluid technology were studied when extracting the total alkaloids from lotus leaf. The extraction parameters such as extraction temperature, extraction pressure, co-solvent flow rate and extraction time were optimized based on single-factor and orthogonal experiment. And the optimum extracting conditions were as follows:extraction temperature 55℃, extraction pressure 20 MPa, co-solvent flow rate 0.2 mL/min and extraction time 2 h. The sequence of the factors affecting the extraction rate was: extraction temperature>extraction time>extraction pressure>co-solvent flow rate. Under the optimal conditions, the extraction yield of total alkaloids from lotus leaf could reach 318.45μg/g.(2) Ultrasonic-assisted extraction of total alkaloids from lotus leaf was investigated. Based on the single factor experiments, the effects of liquid-solid ratio, ultrasonic power and ethanol concentration on enhancing the extraction yield of total alkaloids from lotus leaf were optimized with Response Surface Methodology (RSM). A 4-factor,3-level Box-Behnken experimental design was applied and the interactions of each factor on the total alkaloids yield were also investigated. The optimal conditions were as follows:liquid-solid ratio 43 mL/g, ultrasonic power 410 W and ethanol concentration 76%. Under such conditions, the total alkaloids yield could reach 326.08μg/g.(3 High-speed countercurrent chromatography (HSCCC) was used to separate the total alkaloids of lotus leaf. A two-phase solvent consisted of n-hexane-ethyl acetate-methanol-water at a volumn rate of 5:5:6:4 was employed. Then, component two and three were further isolated through preparation liquid to obtain four alkaloid monomers. The purities of the four monomers were 91.25%,96%,97.13% and 99.3%, respectively, assayed by HPLC. Then after the chemical structure identification technology through UV and LC/MS, the four monomers were identified to be asimilobine, O-nornuciferine, N-nornuciferine and nuciferine.(4) Oxford cup method was applied to evaluate the antimicrobial activity of total alkaloids and four monomers of lotus leaf. Results showed that they were more effective on the selected bacteria(Staphylococcus aureus, Listeria monocytogenes and Escherichia coli) than on the selected fungi(Penicillium expansum and Saccharomyces cerevisiae). The minimum inhibitory concentration (MIC) for fungi tested was more than 400μg/mL, while the MIC for bacteria tested was ranged from 50 to 400μg/mL. O-nornuciferin was found to have the stronggest antimicrobial activity with only 50μg/mL MIC value against Staphylo coccus aureus, which was equal to the positive control chloramphenicol. An analysis of structure-activity relationship (SAR) showed that the substituent in position C-2 and N of aporphine alkaloids might have effect on the antimicrobial activity.