Isolated And Purified Alkaloids Monomers From Lotus Leaf And Structure-activity Studies Of It On The3T3-L1Preadipocytes

Lotus leaf, which has a wide distribution in our country, is the leaf of Nelumbo nucifera Gaertn. A increasing number of people pay their attention to the lotus leaf since it has plenty of bioactive components such as alkaloid, flavonoids, β-carotene, polysaccharides, organic acids, volatile oils, steroids, soaps, etc. According to the literature, the research about lipid-lowering effect of lotus leaf focused on the effect of crude extract of alkaloid on hyperlipidemia rats. Meanwhile, the effect of lotus leaf extract on3T3-L1preadipocyte has rarely mentioned. With the living standard improving, massive number of obese children is drawing more and more concern and it is significance to develop healthy and green lipid-lowering diet food or drugs. This paper focus on the lotus leaf and discusses the effect of its alkaloid extract and the monomers on3T3-L1preadipocyte cell proliferation, cell cycle, apoptosis and differentiation. Furthermore, the active ingredients of lotus leaves about obesity prevention have also been investigated. The results play a vital role in the development of diet food or drugs. Results showed that:(1)Acidic ethanol extraction method was applied to obtain the crude alkaloids extract of lotus leaf (LLAE).Then measured the effcets of alkaloids extract from lotus leaf on3T3-L1preadipocytes by detecting of cell proliferation, cell cycle, cell apoptosis and differentiation. Results showed that the crude alkaloids extract from lotus leaf (LLAE) can effectively inhibit the proliferation of3T3-L1preadipocytes in a dose and time dependent manner. The IC50values of3T3-L1preadipocytes treated with LLAE for24,48,72hours were171μg/mL,153μg/mL and129μg/mL respectively. LLAE could significantly change cell distribution of3T3-L1preadipocytes. Results showed cell cycles of3T3-L1preadipocytes were arrested in G0/G1phase respectively. PI/Annexin-V FITC staining indicated that LLAE could induce apoptosis of3T3-L1preadipocytes in a dose and time dependent manner. The differentiation of3T3-L1preadipocytes into adipocytes was suppressed with the treatment with LLAE and reduced the accumulation of fat in adipocytes.100μg/mL of LLAE reduced the lipid content in adipocyte by36.7%respectively, compared with the control(P<0.01).(2)The Pre-HPLC was applied to separate and prepare the crude alkaloids extract of lotus leaf,and six monomers was obtained. By mass spectrometry, thin layer chromatography, UV and analytical HPLC identification and control literature,we can speculation that the six monomers were pronuciferine, armepavine, asimilobine, O-nornuciferine, N-nornuciferine, nuciferine,and the purity of them were91.9%,93.8%,83.5%,96.7%,91.8%,94.2%. The use of Pre-HPLC to separate lotus leaf alkaloid extract, preparation conditions as the mobile phase of acetonitrile and0.05%ammonia ratio of48:52, the injection concentration of10mg/mL, the injection volume2mL/second, the flow rate of8mL/min, the detection wavelength was270nm. Under these conditions,we can prepared four alkaloids monomers,they were asimilobine, O-nomuciferine, N-nornuciferine, nuciferine. By second preparation, the conditions for the mobile phase of acetonitrile and0.05%ammonia ratio of28:72, the injection concentration of10mg/mL, the injection volume2mL/second, the flow rate of8mL/min, the detection wavelength was270nm, under this condition, pronuciferine and armepavine were obtained.(3)Measured the effects of six alkaloid monomers on3T3-L1preadipocytes, by detecting of cell proliferation, cell cycle, cell apoptosis and differentiation, and further analysis of its structure effect relationship. Results showed that the inhibition effect on cell proliferation were N-nomuciferine> asimilobine> O-nornuciferine> nuciferine.while pronuciferine and armepavine had no effects. The study on cell distribution showed that cell cycles of3T3-L1preadipocytes were arrested in G2/M phase respectively by asimilobine, in G0/G1phase respectively by O-nornuciferine, in S phase respectively by N-nornuciferin or nuciferine. By comparison the apoptosis-promoting of four kinds of monomers on3T3-L1preadipocytes, from high to low were N-nomuciferine, asimilobine, O-nomuciferine, nuciferine,the effect is almost similar to the inhibition of cell proliferation. Compared the inhibition effect of cell differentiation between N-nornuciferine and nuciferine,we can find that nuciferine had a higher inhibitory effect. Meanwhile, nuciferine can inhibit the differentiation at a low concentration, but the inhibition of proliferation and induce apoptosis of nuciferine were both weak, suggesting that differentiation of preadipocytes may be have no link between proliferation and apoptosis.