| Nelumbo nucifera is the dried young leaf and radicle in the mature seeds of lotus(Nelumbo nucifera).It is a traditional Chinese medicine and has the functions of clearing the heart and calming the mind,reducing blood lipid,lowering blood pressure and so on.The important active components of lotus seed alkaloids are isoquinoline and aporphine alkaloids.Pharmacological studies have shown that these alkaloids have the activities of lowering blood pressure,lowering blood lipids,antioxidation,anti-tumor and so on.In particular,its role in the regulation of fat metabolism has aroused widespread concern,but its application is seriously limited because of the difficulties in the preparation of alkaloids,low oral bioavailability and unclear mechanism of action.The purpose of this study is to establish a process for the extraction,separation and purification of alkaloids from lotus seed,and on this basis,to construct the drug loading system of lotus seed alkaloid nanoself-microemulsion to improve its oral bioavailability and prolong its action time.Its hypolipidemic activity and mechanism were explored through cell and animal experiments in vivo,so as to provide an effective guarantee for its clinical application.The main contents and results are as follows:(1)Study on the extraction technology of alkaloids from lotus seed: the identification methods of alkaloids from lotus seed by silica gel thin layer chromatography,high performance liquid chromatography and ultraviolet spectrophotometry were established,and the methodology was systematically investigated.Acid water was used to extract alkaloids from lotus seed.Firstly,the extraction efficiency of alkaloids from lotus seed was investigated by different p H,different ratio of material to liquid,extraction time and extraction times.On the basis of single factor experiment,the extraction process of alkaloids from lotus seed was optimized by orthogonal experiment.The experimental results show that the alkaloids in lotus seed can be extracted more fully by using hydrochloric acid aqueous solution.The optimum extraction conditions are as follows: p H value of extraction solution is 2.0,the ratio of material to liquid is 1:30,extraction time is 12 h,extraction times are 3 times.The yield of total alkaloids and Neferine were 9.88% and 1.11%,respectively.The results of methodological experiments show that the precision,repeatability and stability of the results are good,the recovery rate of adding samples is high,and the method is feasible.(2)Study on the separation and purification of alkaloids from lotus seeds: the total alkaloids from lotus seeds were obtained by organic solvent extraction,and the alkaloids were separated and purified by macroporous adsorption resin combined with preparative liquid chromatography.To compare the extraction efficiency of lotus seed alkaloids by dichloromethane and ethyl acetate,to study the adsorption and desorption characteristics of AB-8,DM-130,LX60 and polyamide resins with four different polarity resins,and to optimize the best resin type.Different concentrations of methanol solution as eluent,different sample volume and different flow rate were investigated respectively.The prepared liquid chromatographic separation of lotus seed alkaloid monomer compounds was compared,and the best chromatographic method was selected.The experimental results show that the extraction rate of dichloromethane is better than that of ethyl acetate,and the yield is 7.58%.The enrichment of lotus seed alkaloids by macroporous adsorption resin is better,the adsorption rate is 84.61%,and the desorption rate is 70.11%.Using preparative liquid chromatography,the mobile phase was methanol-water(80-20),the column temperature was greenhouse,the flow rate was 6.0 ml/min,the sample volume on the column was 100.0 mg,and the collection time was 180 min.Four kinds of single lotus seed alkaloids could be obtained quickly and efficiently,and the ratio of peak area detected by HPLC was more than 98%.(3)To study and establish the construction method of lotus seed alkaloid nano-selfmicroemulsion: firstly,the solubility of lotus seed alkaloid in different oil phase,different emulsifier and co-emulsifier was determined,and the pseudo-ternary phase diagram was used to optimize the best oil phase,emulsifier and co-emulsifier.The prescription was further optimized on the basis of pseudo-ternary phase diagram.Taking the mass of oil phase(X1),the mass ratio of emulsifier to co-emulsifier Km(X2)as independent factors,the particle size(Y1),Zeta potential(Y2)and drug loading(Y3)of nano-emulsion as the investigation index,and the total evaluation OD value as the effect variable,two-factor and five-level star design was carried out by using Design-Expert8 statistical software,and the best excipients composition and ratio were optimized.The experimental results show that the optimal prescription of total alkaloid self-microemulsion of lotus seed is ethyl oleate(15%)and RH40-PEG400(2%).The maximum drug loading of this prescription is about8%,and the particle size of the emulsion is about 20 nm Zeta potential 11 m V,which is a stable drug loading system of nano-self-microemulsion.(4)The effect of alkaloids of lotus seed on the differentiation of 3T3-L1 preadipocytes:firstly,the toxicity of total alkaloids and monomer compounds of lotus seeds to 3T3-L1 preadipocytes was determined by MTT method.The differentiation of 3T3-L1 cells was detected by oil red O staining,the lipid content in 3T3-L1 cells was detected by TG kit,and the protein expression was detected by Western blot method to study the effect of lotus seed alkaloids on fat metabolism of 3T3-L1 preadipocytes.The results showed that compared with the model group,the alkaloids of lotus seed could effectively reduce the content of triglycerides in 3T3-L1 cells,including 42.3% of total alkaloids,14.5% of Liensinine,14.3%of Isoliensinine and 14.9% of Neferine.At the same time,the alkaloids of lotus seed could reduce the expression of PPARγ and C/EBPβ protein and inhibit the formation of fat.(5)preliminary pharmacokinetic study of Neferine and Neferine nano-selfmicroemulsion in rats: two groups of SD rats were orally administered with neferine nanomicroemulsion and Neferine carboxymethyl cellulose sodium suspension at a dose of 200mg/kg,and 0.5 m L was collected from rat orbit at 0.25,0.5,0.75,1,1.5,2,3,4,6 and 10 h after administration,respectively.the results showed that Neferine nano-self-microemulsion and Neferine nano-self-microemulsion were administered orally to two groups of Neferine nano-self-microemulsion and Neferine carboxymethyl cellulose sodium suspension at a dose of 200 mg/kg,respectively.The plasma concentration of Neferine in different time was analyzed by high performance liquid chromatography(HPLC).The maximum plasma concentration and peak time of Neferine were compared,and the absorption and elimination of Neferine in nano-self-microemulsion and non-nano-self-microemulsion were determined.The experimental results showed that Nef was absorbed in the blood of rats,and the maximum blood concentration and peak time of Nef treated with nano-self-microemulsion were higher than those of non-nano-Nef,the maximum blood concentration increased by27.32%,and the peak time was 0.25 h faster.Nef treated by nano-treatment effectively improved its bioavailability.In this study,the process of extraction,separation and purification of alkaloids from lotus seed was established,and the drug loading system of nano-self-microemulsion was successfully constructed.3T3-L1 cell experiment in vitro showed that alkaloids from lotus seed could effectively reduce the accumulation of intracellular fat,the level of triglyceride and the expression of PPARγ and C/EBPβ protein.Pharmacokinetic studies showed that the drug loading system of nano-self-microemulsion could significantly increase the maximum blood concentration and peak time of alkaloids in lotus seed,improve its bioavailability.The results of this study provide a theoretical basis and technical guarantee for the clinical application of lotus seed and its alkaloids. |
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